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Cytokine mRNA expression in intestinal tissue of interleukin-2 deficient mice with bowel inflammation  

PubMed Central

Background—Mice deficient in interleukin-2 (IL-2) develop inflammatory bowel disease resembling ulcerative colitis in humans. Recent studies provided evidence that ?? T cells, particularly CD4 T cells, rather than B cells, are involved in the pathogenesis of bowel inflammation of IL-2 deficient mice. ?Aim—To analyse the pattern of expression of cytokine mRNA in intestinal tissue of normal and IL-2 deficient mice. ?Methods—Expression of ?-actin, IL-1?, IL-1?, IL-6, IL-10, tumour necrosis factor ? (TNF-?), interferon ? (IFN-?) and transforming growth factor ?1 (TGF-?1) mRNA was analysed in colon and small intestinal tissue of both IL-2 deficient (IL-2?/?) mice and normal (wild type) litter mates (IL-2+/+) at different ages by using qualitative, as well as semiquantitative, competitive reverse transcription polymerase chain reaction (RT-PCR). Results were correlated with the phase of progression of the disease, as determined by histology. ?Results—IL-2?/? mice had expressed low levels of IL-1?, IL-1?, IL-6, TNF-?, and IFN-? mRNA in the colon by 1.5 weeks of age. In advance of the development of histologically and clinically detectable bowel inflammation, expression of IL-1?, IL-1?, IL-6, TNF-?, IFN-?, and IL-10, but not TGF-?1, mRNA increased in the colon of IL-2 deficient mice. In contrast, IL-2+/+ mice expressed TGF-?1 mRNA in colon tissue at 13 and 23 weeks of age, but not IL-1?, IL-1?, IL-6, TNF-?, IL-10, or IFN-? mRNA. Levels of expression of cytokine mRNA in tissue from the small intestine were comparable in IL-2?/? and IL-2+/+ mice. ?Conclusions—Bowel inflammation in IL-2 deficient mice is preceded by an increase in IL-1?, IL-1?, TNF-?, and IFN-? mRNA expression in colon tissue. Low levels of TGF-?1, but high levels of IL-1?, IL-1?, IL-6, TNF-?, IFN-?, and IL-10 mRNA expression correlate with the manifestation of severe colitis, and suggest that T cells and macrophages are involved in bowel inflammation of IL-2 deficient mice. ?? Keywords: cytokine; mRNA expression; interleukin-2 deficient mice; bowel inflammation

Autenrieth, I; Bucheler, N; Bohn, E; Heinze, G; Horak, I



Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA.  


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

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



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

PubMed Central

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

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



Avian Interleukin-2.  

National Technical Information Service (NTIS)

A substantially pure species of avian Interleukin-2 has a molecular weight of about 14 kda. as determined by high resolution gel filtration chromatography. The compound is obtained from avian lymphocytes. It is produced by collecting lymphocytes from an a...

T. L. Fredericksen J. M. Sharma



Avian Interleukin-2.  

National Technical Information Service (NTIS)

A substantially pure species of avian Interleukin-2 has a molecular weight of about 30 kda. as determined by SDS-polyacrylamide gel electrophoresis. The compound is obtained from avian lymphocytes. It is produced by collecting lymphocytes from an avian do...

T. L. Fredericksen J. M. Sharma



Coexpression of an unusual form of the EWS–WT1 fusion transcript and interleukin 2/15 receptor ?mRNA in a desmoplastic small round cell tumour  

PubMed Central

Background The ? chain of the interleukin 2/15 receptor (IL?2/15R?) is induced by the expression of the EWS–WT1. A case of desmoplastic small round cell tumour (DSRCT) expressing only an unusual EWS?WT1 treated by us is reported here. Aim To characterise an unusual form of EWS–WT1. Methods Frozen tissue sections of the axillary tumour were examined using a laser?assisted microdissection technique and reverse transcriptase polymerase chain reaction. Results The novel fusion of exon 8 of EWS and the defective exon 10 of WT1 (?KTS) was detected. Although it was an unusual form, the coexpression of the present EWS–WT1, IL?2/15R? and Janus kinase (JAK1) mRNA was detected in the tumour cells. IL?2 and signal transducers and activators of transcription (STAT5) mRNA were detected in both tumour and stromal cells. Conclusion The induction of the IL?2/15 receptor signalling pathway may contribute to tumorigenesis in DSRCT through a paracrine or an autocrine system, even though the EWS–WT1 was an unusual form.

Nakanishi, Y; Oinuma, T; Sano, M; Fuchinoue, F; Komatsu, K; Seki, T; Obana, Y; Tabata, M; Kikuchi, K; Shimamura, M; Ohmori, K; Nemoto, N



Interleukin-2 in autologous bone marrow transplantation.  


Interleukin-2 results in the generation of lymphokine activated killer cells which exhibit a potent effect against a wide variety of tumours. Consequently, interleukin-2 therapy has been used to induce a graft versus tumour effect following autologous bone marrow transplantation. Preclinical studies have shown that this results in successful engraftment, and an enhanced reconstitution of the immune system. PMID:8453356

Charak, B S


Bacteriolytic activity of human interleukin-2.  


In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme - chicken egg white lysozyme - by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases. PMID:23240569

Levashov, P A; Sedov, S A; Belogurova, N G; Shipovskov, S V; Levashov, A V



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

PubMed Central

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 45?. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-? 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.

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



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


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 1mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G(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 45?. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-? or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. PMID:22285274

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



Suppression of MMP-2 Attenuates TNF-? Induced NF-?B Activation and Leads to JNK Mediated Cell Death in Glioma  

PubMed Central

Background Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines. Methodology/Principal Findings MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-? with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKK? and pI?B? expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-?, TRADD, IKK? and pI?B? expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-?B activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections. Conclusion/Significance In summary, our study implies a role of MMP-2 in the regulation of TNF-? mediated constitutive NF-?B activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo.

Kesanakurti, Divya; Chetty, Chandramu; Bhoopathi, Praveen; Lakka, Sajani S.; Gorantla, Bharathi; Tsung, Andrew J.; Rao, Jasti S.



A viral long terminal repeat in the interleukin 2 gene of a cell line that constitutively produces interleukin 2.  

PubMed Central

The gibbon leukemia cell line MLA 144 differs from every other T-lymphocyte line in that it constitutively makes interleukin 2 (IL-2) (also called T-cell growth factor) without stimulation by antigen, lectin, or tumor promoters. Previous work in which glucocorticoids were used to inhibit IL-2 production has indicated that proliferation of this cell line is dependent upon endogenously produced IL-2. We have found that the MLA 144 cell line has a copy of the gibbon leukemia virus inserted into the 3' nontranslated region of the IL-2 gene. This integration event produces a composite mRNA made up of the protein coding sequences of the IL-2 gene transcript but incorporating the viral long terminal repeat (LTR) in the 3' nontranslated region of the mRNA. This composite mRNA transcript uses the polyadenylylation signal in the viral 5' LTR and incorporates the viral transcriptional control regions. The integration event must involve only one allele of the IL-2 gene, since transcripts essentially identical to normal human IL-2 mRNA are also produced in cloned sublines of MLA 144. That the viral LTR contains a 94-base-pair repeat reminiscent of enhancer sequences in several viruses suggests that the integration of the viral LTR at the 3' end of the IL-2 gene is responsible for the constitutive production of IL-2 in the MLA 144 cell line. Images

Chen, S J; Holbrook, N J; Mitchell, K F; Vallone, C A; Greengard, J S; Crabtree, G R; Lin, Y



Advances in interleukin 2 receptor targeted treatment  

PubMed Central

T cell activation and cellular immune responses are modulated by interleukin 2 (IL2) through binding to its corresponding cell surface receptor. Three forms of the receptor are recognised based on IL2 binding affinity. The high affinity receptor is a heterotrimer composed of ?, ?, and ?c-polypeptide chains. The 55 kDa ?-chain also known as the Tac (T cell activation) antigen or CD-25 is a unique subunit of the high affinity IL2 receptor (IL2R?). Resting T cells express few IL2R?, however, when activated, the expression of ILR2? rapidly increases. The IL2R? is shed from the cell surface and is measurable in the serum as a 45 kDa soluble form (s-Tac or s-IL2R?). Serum concentrations of s-Tac can be used as a surrogate marker for T cell activation and IL2R? expression. IL2R? is over expressed by T cells in a number of autoimmune diseases, allograft rejection and a variety of lymphoid neoplasms. IL2 induced proliferation of T cells can be inhibited by the murine monoclonal antibody (anti-Tac) directed against the ?-chain of the IL2R. Through molecular engineering, murine anti-Tac has been humanised reducing its immunogenicity without changing its specificity. Humanised anti-Tac (HAT) has been shown to reduce the incidence of renal and cardiac allograft rejection as well as decrease the severity of graft versus host disease in patients undergoing HLA matched allogeneic bone marrow transplantation. IL2R? targeted treatment with radioimmunoconjugates of anti-Tac and immunotoxins has shown promise in the treatment of CD25 expressing lymphomas.??

Morris, J.; Waldmann, T.



Soluble interleukin-2 receptor as a predictor of neonatal sepsis  

Microsoft Academic Search

We prospectively measured soluble interleukin-2 receptor levels in 56 premature infants with suspected sepsis and demonstrated significant differences between those with positive results on blood, urine, or cerebrospinal fluid cultures, and those with negative results. Soluble interleukin-2 receptor levels can be used to facilitate the diagnosis of sepsis in premature infants with negative blood culture results. (J PEDIATR 1995;126:982-5)

Michael L. Spear; John L. Stefano; Paul Fawcett; Roy Proujansky



Snake venom toxin from vipera lebetina turanica induces apoptosis of colon cancer cells via upregulation of ROS- and JNK-mediated death receptor expression  

PubMed Central

Background Abundant research suggested that the cancer cells avoid destruction by the immune system through down-regulation or mutation of death receptors. Therefore, it is very important that finding the agents that increase the death receptors of cancer cells. In this study, we demonstrated that the snake venom toxin from Vipera lebetina turanica induce the apoptosis of colon cancer cells through reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) dependent death receptor (DR4 and DR5) expression. Methods We used cell viability assays, DAPI/TUNEL assays, as well as western blot for detection of apoptosis related proteins and DRs to demonstrate that snake venom toxin-induced apoptosis is DR4 and DR5 dependent. We carried out transient siRNA knockdowns of DR4 and DR5 in colon cancer cells. Results We showed that snake venom toxin inhibited growth of colon cancer cells through induction of apoptosis. We also showed that the expression of DR4 and DR5 was increased by treatment of snake venom toxin. Moreover, knockdown of DR4 or DR5 reversed the effect of snake venom toxin. Snake venom toxin also induced JNK phosphorylation and ROS generation, however, pretreatment of JNK inhibitor and ROS scavenger reversed the inhibitory effect of snake venom toxin on cancer cell proliferation, and reduced the snake venom toxin-induced upregulation of DR4 and DR5 expression. Conclusions Our results indicated that snake venom toxin could inhibit human colon cancer cell growth, and these effects may be related to ROS and JNK mediated activation of death receptor (DR4 and DR5) signals.



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

SciTech Connect

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.

Latchoumycandane, Calivarathan [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Seah, Quee Ming [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Tan, Rachel C.H. [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Sattabongkot, Jetsumon [Armed Forces Research Institute of Medical Sciences, Bangkok 10400 (Thailand); Beerheide, Walter [Siam Life Science Ltd., Bangkok 10500 (Thailand); Boelsterli, Urs A. [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore) and Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117597 (Singapore)]. E-mail:



JNK-mediated Phosphorylation of Paxillin in Adhesion Assembly and Tension-induced Cell Death by the Adenovirus Death Factor E4orf4*S?  

PubMed Central

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.

Smadja-Lamere, Nicolas; Boulanger, Marie-Chloe; Champagne, Claudia; Branton, Philip E.; Lavoie, Josee N.



Qualitative and quantitative evaluation of a local lymph node assay based on ex vivo interleukin-2 production  

Microsoft Academic Search

The local lymph node assay (LLNA) is a regular method for the detection of sensitizing chemicals in mice which measures the incorporation of tritiated thymidine in lymph node cells. We have evaluated an alternative to this method based on the interleukin-2 (IL-2) production of lymph node cells.At the mRNA level, no change in the IL-2 gene expression level was detected

Philippe Azam; Jean-Luc Peiffer; Jean-Claude Ourlin; Pierre-Antoine Bonnet; Marie-Hélène Tissier; Laurence Vian; Isabelle Fabre



Recent Advances in the Understanding of Interleukin2 Signal Transduction  

Microsoft Academic Search

Interleukin-2 is one of the critical cytokines that control the proliferation and differentiation of cells of the immune system. The present article briefly reviews the current and recently established knowledge on the intracellular signaling events that convert the initial interaction of IL-2 with its receptor into pathways leading to the various biological functions. A first step in IL-2 signaling is

Franck Gesbert; Maryvonnick Delespine-Carmagnat; Jacques Bertoglio



Interleukin-2 Signal Transduction: A Diffusion-Kinetics Model.  

National Technical Information Service (NTIS)

The diffusion-kinetics model for the interactions between interleukin-2 and each of its T-cell surface receptors (IL-2 alpha and IL-2 beta) is presented. This model is unique in that it considers both three dimensional ligand-receptor interactions and two...

M. P. Keith



Interleukin2 Binds to Ganglioside GD 1b  

Microsoft Academic Search

We have developed a solid matrix immunoassay to determine the binding of interleukin-2 (IL-2) to specific gangliosides. The assay establishes that recombinant human IL-2 binds to ganglioside GD1b but not to any other gangliosides (GM1, GM2, GM3, GD1a, GD2, GD3, and GT1b). The binding varies with the ratio of GD1b and IL-2. This assay enables distinguishing the nature of the

Mepur H. Ravindranath; Alexandra Gonzales; Daniel Soh; Kevin Nishimoto; Wai-Yin Tam; Anton Bilchik; Donald L. Morton; Steven O'Day



Adenovirus-mediated interleukin-2 gene therapy of nociception  

Microsoft Academic Search

The effect of adenovirus-mediated interleukin-2 (IL-2) gene on rat basal nociceptive response and chronic neuropathic pain was explored. The paw withdrawal latency induced by radiant heat was used to evaluate the antinociceptive effect of adenovirus type 5 (Ad5) and Ad5-IL-2. The results showed that intrathecal delivery of Ad5-IL-2 exhibited obvious antinociceptive effects on basal nociceptive response and chronic neuropathic pain,

M Z Yao; J F Gu; J H Wang; L Y Sun; H Liu; X Y Liu



Interleukin-2 Encapsulated in and/or Bound to a Carrier Erythrocyte.  

National Technical Information Service (NTIS)

A method and composition are provided by which interleukin-2 is encapsulated in and/or bound to erythrocytes and the thus encapsulated and/or bound interleukin-2 is injected for therapeutic purposes. Interleukin-2 has been encapsulated in and/or bound to ...

J. R. DeLoach K. Andrews C. L. Sheffield K. E. Koths



Bone marrow transplantation with interleukin-2-activated bone marrow followed by interleukin-2 therapy for acute myeloid leukemia in mice.  


We have investigated approaches to induce graft-versus-leukemia (GVL) effect in autologous bone marrow transplantation (ABMT) without graft-versus-host disease to improve survival and cure in leukemia. The present study shows that bone marrow transplantation (BMT) using syngeneic bone marrow activated with interleukin-2 (ABM) for 24 hours in vitro, followed by interleukin-2 (IL-2) therapy, was superior to BMT with fresh, syngeneic bone marrow (FBM) in terms of survival and cure in mice with acute myeloid leukemia (P less than .001) and led to normal hematopoietic reconstitution. Addition of IL-2 therapy after BMT with FBM did not improve the results over BMT with FBM alone (P = .98). These results suggest that the GVL effect of ABMT can be enhanced by using ABM for BMT followed by IL-2 therapy without compromising engraftment. PMID:2257292

Charak, B S; Brynes, R K; Groshen, S; Chen, S C; Mazumder, A



[Immunomodulating properties of synthetic fragments of human interleukin-2].  


For the study of the antigenic structure and function of human interleukin 2, the peptides corresponding to its 60-72 sequence were synthesized by conventional methods of peptide chemistry in solution. To enhance the stability of the synthetic peptides towards the proteolysis and to remove their terminal charges, we acetylated their NH2 groups and esterified with methanol their carboxyls. Some of these peptides were converted from being cytotoxic to possessing strong growth-stimulating activity for the preliminary activated macrophages both in vitro and in vivo. The biologically active peptides were also shown to enhance regeneration-reparation processes in liver and skin. PMID:8687510

Onoprienko, L V; Mikhaleva, I I; Ivanova, V T; Vo?tenkov, B O; Okulov, V B



Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.  

PubMed Central

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H



Expression of porcine interleukin-2 in Escherichia coli.  


A mature form of porcine interleukin-2 (IL-2) protein without signal peptides was expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli using pGEX vector. Since most of GST-IL-2 fusion protein was detected in an insoluble fraction on SDS-PAGE analysis, the insoluble fusion protein was solubilized by refolding procedure using urea. The recombinant IL-2 (rIL-2) was purified by a batch method using Glutathione Sepharose 4B and factor Xa digestion and used for preparation of antisera in mice. The antisera reacted with rIL-2 expressed in baculovirus system on immunoblot analysis. In addition, the purified rIL-2 showed a high biological activity on CTLL-2 proliferative response. PMID:11073083

Iwata, H; Yamamoto, M; Hasegawa, A; Kurata, K; Inoue, T



Secretion of biologically active porcine interleukin-2 by Lactococcus lactis.  


In this study, secretion of two functional recombinant porcine interleukin-2 (rIL-2) proteins by Lactococcus lactis was studied. Two secretion cassettes were constructed in which the secretion was achieved by gene fusion between the lactococcal usp45 secretion signal, a synthetic propeptide and the sequence encoding the mature IL-2. In addition, one of the two secretion cassettes contained the H-domains of L. lactis PrtP. Both of the constructed recombinant IL-2 proteins were found to be secreted in the same quantities, approximately 0.5mg/l. According to a cell proliferative assay using CTLL-2 cell line the specific biological activities of both purified rIL-2 proteins were found to be of similar levels. PMID:16549279

Avall-Jääskeläinen, Silja; Palva, Airi



Expression of functional interleukin 2 receptors on chronic lymphocytic leukaemia B lymphocytes is modulated by recombinant interleukin 2.  

PubMed Central

In 16 of 18 chronic lymphocytic leukaemia (CLL) patients examined, a significant proportion of B cells in the leukaemic clone bound monoclonal antibodies specific for the interleukin 2 (IL-2) receptor site (CD25). B lymphocytes from patients tested showed a direct response to recombinant interleukin (rIL-2) during culture in vitro as shown by: (a) a ligand-mediated upregulation in the level of IL-2 receptor (IL-2R) expression (12 of 12 patients), (b) an increase in cell size (eight of nine patients), (c) an increase in 3H-thymidine uptake (four of six patients). Taken together, this evidence suggests that the majority of leukaemic B cells from all the CLL patients examined expressed functional IL-2 receptors in vitro. Intriguingly, maximal receptor upregulation or increase in cell size was achieved at a lower concentration (50 u/ml) of rIL-2 than was required to achieve maximal 3H-thymidine incorporation.

Murphy, J J; Malkovska, V; Hudson, L; Millard, R E



Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.  

PubMed Central

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel. Images

Devos, R; Plaetinck, G; Cheroutre, H; Simons, G; Degrave, W; Tavernier, J; Remaut, E; Fiers, W



Concanavalin A-inducible, interleukin-2-producing T cell hybridoma  

PubMed Central

The fusion of an AKR T cell tumor line to normal B6D2F1, T cells resulted in the production of a cloned T cell hybridoma (FS6-14.13) inducible with the mitogen concanavalin A (Con A). The supernate from Con A-stimulated hybridoma cells was active both in the stimulation of an anti-sheep red blood cell response by partially T cell-depleted B cells and in the stimulation of the growth of antigen-specific T cell blasts. The active principle in both assays had a molecular weight of approximately 30-40,000. These results indicated the presence of interleukin 2 (IL2) in the hybridoma supernate. The activity of the hybridoma supernate in B cell responses was dependent on the presence of adherent cells and a few contaminating T cells. On the other hand, Con A-stimulated supernates from normal spleen cells were active after either adherent cell removal or severe T cell depletion. These results suggested that IL2 was the only active helper factor in the hybridoma supernate, but that additional helper factors were present in supernates from Con A-stimulated normal spleen cells.



Porcine interleukin 2: parameters of production and biochemical characterization.  


Interleukin 2 (IL2) or T cell growth factor (TCGF) has been characterized in a number of species but not in porcines. Porcine IL2 was detected in supernates (SN) of cultures of pig lymphocytes by: 1) the stimulation of the IL2-sensitive murine T cell line, CT6; 2) a costimulator assay involving porcine thymocytes; and 3) by the in vitro maintenance of antigen or mitogen-induced porcine lymphoblastoid cells. Porcine IL2 production by pig lymphocytes was induced by the mitogens Concanavalin A (Con A) Phytohemagglutiniin (PHA), and Pokeweed mitogen (PWM), but not by lipopolysaccharide (LPS). IL2 activity was demonstrated in the SN of mitogen-stimulated lymphocyte cultures as early as 24 hr after initiation of culture, reached peak levels at 48 hr, and decreased by 72 hr. Mitogens induced IL2 secretion by pig peripheral blood mononuclear cells, lymph node cells, and spleen cells, but not thymus cells. The cells responsible for IL2 production are presumptive T cells because: 1) they are nylon wool non-adherent; and 2) are non-surface-Ig bearing. In contrast, SN from cultures of surface Ig-positive cells had minimal IL2 activity. Porcine IL2 resembles rat and human IL2 in that it has an apparent molecular weight of approximately 15,000, and does not bind to DEAE-cellulose (DE-52) ion exchange columns equilibrated in 0.05 M sodium phosphate buffer (pH 7.6). PMID:6609481

Gasbarre, L C; Urban, J F; Romanowski, R D



Binding and internalization of biotinylated interleukin-2 in human lymphocytes.  


The binding, internalization, and fate of interleukin-2 (IL-2) were studied in phytohemagglutinin (PHA)-activated human lymphocytes using biotinylated recombinant IL-2 (rIL-2). Streptavidin adsorbed to 18-nm colloidal gold beads (Au18-streptavidin) and streptavidin covalently bound to horseradish peroxidase (HRP-streptavidin) were used to follow the movement of biotinylated rIL-2 within cells over a 4-hour period. Results obtained from either probe were similar. Biotinylated rIL-2 was taken up in coated pits, transferred to a series of small uncoated vesicles and tubules in the peripheral cytoplasm of the cell, then concentrated and sequestered in uncoated vesicles, multivesicular bodies (MVB), and dense bodies (DB) in the peripheral and juxtanuclear cytoplasm of the cell. Occasionally, MVB containing Au18-streptavidin, or HRP-streptavidin, appear to have fused with the plasma membrane of the cell. No labeling of the Golgi cisternae, nuclear envelope, or nucleus was observed. Results from a competitive receptor binding assay and a cell proliferation assay indicate that both the affinity of rIL-2 for high affinity rIL-2 receptors and the proliferative activity of rIL-2 were negligibly affected by the biotinylation procedure. These studies suggest that in activated lymphocytes, IL-2 is bound to receptors on the cell surface, gathered in coated pits, internalized by receptor-mediated endocytosis, concentrated in the endosomal compartments, and delivered to lysosomes for degradation. PMID:2163697

Peters, D K; Norback, D H



[Interleukin-2 (IL-2) in active pulmonary tuberculosis].  


To clarify the precise of cellular immunity mechanism in pulmonary tuberculosis, we investigated the amount of IL-2 in patients with untreated active pulmonary tuberculosis. When serum adenosine deaminase (ADA) activity was examined using enzyme assay, an abnormally high level was observed in all patients (29.0 + 11.6 IU/ml, mean + SD; 4.5-17.8, normal range). Likewise, the level of serum-soluble interleukin-2 receptor (IL-2R) measured by ELISA showed abnormal high level in all patients (844.3 + 584.8 IU/ml; 80-300, normal range). When stimulated using PHA, the peripheral lymphocyte's ability to produce IL-2 revealed no difference between control subjects and patients. It was, however, noted that the lymphocytes of the patients significantly suppressed IL-2 responsiveness when compared to the control subjects (P less than 0.05). The serum IL-2 concentration measured using RIA could not be detected in any of the patients as was the same for control subject. All of the above mentioned results suggest that T-cell activation which caused increment in serum ADA activity and soluble IL-2R occurred in active pulmonary tuberculosis. The suppressed IL-2 responsiveness in the peripheral lymphocytes of patients proposes the possibility of soluble IL-2R reduction by the negative feedback mechanism in IL-2-sensitive lymphocytes. PMID:1766152

Ida, T; Taniai, S; Makiguchi, K; Otomo, N; Taniguchi, K; Miyazato, I; Chida, M; Ichioka, M; Marumo, F



Recombinant interleukin 2 therapy in severe combined immunodeficiency disease.  

PubMed Central

Severe combined immunodeficiency disease (SCID) is a congenital disorder of severe B- and T-lymphocyte dysfunction in which several pathogenic mechanisms have been identified. The present study describes a female child with SCID who had a primary defect in the ability of T cells to secrete interleukin 2 (IL-2). B- and T-cell numbers were normal, but their functions were severely deficient. Mitogen and antigen-driven lymphoproliferative responses were diminished but were correctable in vitro with recombinant IL-2 (rIL-2). The patient's phytohemagglutinin-stimulated lymphocytes expressed IL-2 receptors normally. Despite the presence of the gene for IL-2, the patient's cells were grossly deficient in messenger RNA for IL-2 and endogenous IL-2 production. Pokeweed mitogen-driven B-cell differentiation was decreased and was not corrected by the addition of normal T cells to the B cells. Two attempts at immune reconstitution by haploidentical bone marrow transplantation failed. Therapy with rIL-2 (30,000 units/kg, given daily i.v.) resulted in marked clinical improvement as well in improved T-cell functions. The child, now 3 yr old, has been on rIL-2 therapy for 2 yr and receives rIL-2 (30,000 units/kg) three times weekly at home. This case study points to a new direction in the treatment of such disorders with rIL-2. Images

Pahwa, R; Chatila, T; Pahwa, S; Paradise, C; Day, N K; Geha, R; Schwartz, S A; Slade, H; Oyaizu, N; Good, R A



Interleukin-2 in bone marrow transplantation: preclinical studies.  


Interleukin-2 (IL-2) promotes the generation and proliferation of killer cells in the peripheral blood and bone marrow (BM) both in vitro and in vivo. When employed in a syngeneic bone marrow transplantation (BMT) setting and followed by IL-2 therapy, murine BM cells activated with IL-2 in vitro (ABM) demonstrate potent graft-versus-leukemia (GVL) and anticytomegalovirus effects. ABM cells retain the capacity to reconstitute the hemopoietic system both in normal and leukemic mice. This therapy does not cause graft-versus-host disease (GVHD). Human ABM cells carry out purging of leukemia without loss of progenitor cell activity in vitro. The purging ability of ABM can be augmented by interleukin-1, interferon, and tumor necrosis factor. IL-2 therapy stimulates the veto suppressor cell activity of T cell-depleted BM, and has reduced GVHD and permitted engraftment of mismatched allogeneic BM in murine models. Future studies should determine the optimum treatment schedules with IL-2 for improving the GVL effect in autologous BMT, and for abolishing GVHD in allogeneic BMT settings. PMID:1326364

Charak, B S; Choudhary, G D; Tefft, M; Mazumder, A



Contribution of a p75 interleukin 2 binding peptide to a high-affinity interleukin 2 receptor complex  

SciTech Connect

There are at least two forms of cellular receptors for interleukin 2(IL-2); one with a very high affinity and the other with a lower affinity. The authors identified a non-Tac IL-2 binding peptide with a relative molecular weight of 75,000 (p75). Cell lines bearing either the p55 Tac or the p75 peptide alone manifested low-affinity IL-2 binding, whereas a cell line bearing both peptides manifested both high- and low-affinity receptors. Cross-linking studies were performed with /sup 125/I-labeled IL-2. After the internalization of labeled IL-2 through high-affinity receptors, the p75 peptide could not be detected by cross-linking studies. Furthermore, fusion of cell membranes from low-affinity IL-2 binding cell lines bearing the Tac peptide alone with membranes from a cell line bearing the p75 peptide alone generated hybrid membranes bearing high-affinity receptors. These results suggest a multichain model for the high-affinity Il-2 receptor in which high-affinity receptors would be expressed when both Tac and p75 Il-2 binding peptides are present and associated in a receptor complex.

Tsudo, M.; Kozak, R.W.; Goldman, C.K.; Waldmann, T.A.



Myelostimulatory activity of recombinant human interleukin-2 in mice  

SciTech Connect

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.

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



Arf6-GEF BRAG1 regulates JNK-mediated synaptic removal of GluA1-containing AMPA receptors: a new mechanism for nonsyndromic X-linked mental disorder.  


Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disabilities. BRAG1 is a guanine nucleotide exchange factor for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif; however, the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors, depresses AMPA receptor (AMPA-R)-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic XLID. PMID:22915114

Myers, Kenneth R; Wang, Guangfu; Sheng, Yanghui; Conger, Kathryn K; Casanova, James E; Zhu, J Julius



The Toxicity of Recombinant Human Interleukin-2 in Rats following Intravenous Infusion  

PubMed Central

Studies of recombinant interleukin-2 (RIL-2) administered by continuous intravenous infusion revealed hepatocellular toxicity and redistribution of lymphoid cells. This finding was different from the normal findings seen in rats receiving comparable infusions of the vehicle.

Matory, Yvedt L.; Chang, Alfred E.; Lipford, Edward H.; Braziel, Rita; Hyatt, Cornelia L.; Rosenberg, Steven A.



Effect of 1,1-Dimethylhydrazine on Lymphoproliferation and Interleukin 2 Immunoregulatory Function.  

National Technical Information Service (NTIS)

The studies reported here suggest that the immunomodulatory effects of 1,1-dimethylhydrazine (UDMH) are associated, in part, with interference with interleukin 2 (IL-2) regulatory action. Concanavalin A (Con A)-stimulated (deoxyribonucleic acids) synthesi...

R. M. Bauer M. J. Tarr R. G. Olsen



Interleukin-2 induces the proliferation of mouse primordial germ cells in vitro.  


Primordial germ cells (PGCs) are the stem cell precursors of the germ line. Several growth factors contribute to enlarging the PGC population by acting as mitogens, survival factors or both. Interleukin-2 (IL-2) has a growth-promoting activity for T and B-lymphocytes, but its role in PGCs had not yet been studied. Here, we show that PGCs isolated from 10.5, 11.5 and 12.5 day postcoitum (dpc) mouse embryos constitutively express the three subunits (alpha, beta and gamma) of the IL-2 receptor (IL-2R). In contrast, IL-2 mRNA was not detected in these cells. However, the addition of recombinant IL-2 to the culture medium increased the number of PGCs in vitro via a mitogenic effect, as indicated by bromodeoxyuridine incorporation assays. Neutralization of the IL-2 receptor using anti-IL-2R subunit antibodies inhibited this IL-2-mediated proliferative effect on PGCs from 11.5 dpc embryos. Together, these data are indicative of a paracrine effect of IL-2 on PGC proliferation. In this regard, we also compared the effect of IL-2 with other compounds such as basic fibroblast growth factor (bFGF), steel factor, leukemia inhibitory factor and forskolin, and found that the degree of proliferation induced by IL-2 was similar to that induced by bFGF and forskolin. These observations support the notion that similar patterns of molecular signaling may underlie the developmental pathways of hematopoietic and germ stem cell precursors. PMID:17939120

Eguizabal, Cristina; Boyano, Maria D; Díez-Torre, Alejandro; Andrade, Ricardo; Andollo, Noelia; De Felici, Massimo; Aréchaga, Juan



Transcriptional Repression of the Interleukin2 Gene by Vitamin D 3 : Direct Inhibition of NFATp\\/AP1 Complex Formation by a Nuclear Hormone Receptor  

Microsoft Academic Search

T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3(1,25(OH)2D3), the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte- macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3




Serum levels of soluble interleukin-2 receptors in acute and chronic viral hepatitis  

Microsoft Academic Search

To analyze interleukin-2-dependent immunoregulatory function in hepatitis B virus infection and in other forms of viral hepatitis, levels of soluble interleukin-2 receptors (sIL-2R) were measured by an enzyme-linked assay in sera from patients with acute and chronic viral hepatitis of different etiology. Increased sIL-2R levels were detected in the early phase of acute hepatitis type A and type B, but

Alfredo Alberti; Liliana Chemello; Giovanna Fattovich; Patrizia Pontisso; Giampietro Semenzato; Cosimo Colletta; Fabrizio Vinante; Giovanni Pizzolo



The p75 Peptide is the Receptor for Interleukin 2 Expressed on Large Granular Lymphocytes and is Responsible for the Interleukin 2 Activation of These Cells  

Microsoft Academic Search

There are at least two interleukin 2 (IL-2) binding peptides: one is the Mr 55,000 peptide (p55) reactive with the anti-Tac monoclonal antibody, and the other is a Mr 75,000 non-Tac IL-2 binding peptide (p75). Independently existing Tac or p75 peptides represent low-affinity IL-2 receptors, whereas high-affinity IL-2 receptors are expressed when both peptides are present and associated in a

Mitsuru Tsudo; Carolyn K. Goldman; Kathleen F. Bongiovanni; Wing C. Chan; Elliott F. Winton; Masato Yagita; Elizabeth A. Grimm; Thomas A. Waldmann



Activation of Interleukin 2 and Interleukin 2 Receptor (Tac) Promoter Expression by the Trans-Activator (tat) Gene Product of Human T-Cell Leukemia Virus, Type I  

Microsoft Academic Search

Cotransfection of cDNA encoding the transactivator gene product of human T-cell leukemia virus, type I (HTLV-I) (tat-I), which acts in trans to augment viral gene expression, has revealed strong regulatory effects of this viral protein on the inducible cellular promoters governing human interleukin 2 (IL-2) and IL-2 receptor (Tac) gene expression. The tat-I protein stimulates a 3- to 6-fold increase

M. Siekevitz; M. B. Feinberg; N. Holbrook; F. Wong-Staal; W. C. Greene



Demonstration of a Non-Tac Peptide that Binds Interleukin 2: A Potential Participant in a Multichain Interleukin 2 Receptor Complex  

Microsoft Academic Search

The interleukin 2 (IL-2) receptor system plays a key role in the T-cell immune response. Although IL-2 binding was reported to be restricted to the Tac peptide, we have identified an IL-2 binding peptide that does not react with anti-human IL-2 receptor monoclonal antibodies, including anti-Tac on MLA 144, a gibbon ape T-cell line. The MLA 144 cell line expressed

Mitsuru Tsudo; Robert W. Kozak; Carolyn K. Goldman; Thomas A. Waldmann



Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.  


Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2. PMID:7553894

Munger, W; DeJoy, S Q; Jeyaseelan, R; Torley, L W; Grabstein, K H; Eisenmann, J; Paxton, R; Cox, T; Wick, M M; Kerwar, S S



Differential Gene Expression in Response to Adjunctive Recombinant Human Interleukin-2 Immunotherapy in Multidrug-Resistant Tuberculosis Patients  

PubMed Central

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-?) 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-? 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 ?-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.

Johnson, Barbara J.; Estrada, Iris; Shen, Zhu; Ress, Stan; Willcox, Paul; Colston, M. Joseph; Kaplan, Gilla



Effect of interleukin-2 and selenium on the growth of squamous cell carcinoma cells  

Microsoft Academic Search

Objectives: The objectives of this study were to determine whether the expression of the interleukin-2 (IL-2) receptors on squamous cell carcinoma cells can be enhanced in the presence of selenium (Se) and contribute to a greater retardation of tumor growth after locoregional therapy with IL-2. Study design: The growth of the cells was studied after in vitro or dietary supplementation

Sarath Thikkurissy; Anthony Pavone; Anthony Rega; Richard Bae; Martin Roy; Harvey I. Wishe; Lidia Kiremidjian-Schumacher



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

Federal Register 2010, 2011, 2012, 2013

...DEPARTMENT OF AGRICULTURE Animal and Plant Health Inspection...Assessment for Field Testing Feline Interleukin-2...Canarypox Vector AGENCY: Animal and Plant Health Inspection...obtain approval from the Animal and Plant Health Inspection...the product for field testing. To determine...



Interleukin 2-regulated in vitro antibody production following a single spinal manipulative treatment in normal subjects  

Microsoft Academic Search

BACKGROUND: Our recent investigations have demonstrated that cell cultures from subjects, who received a single spinal manipulative treatment in the upper thoracic spine, show increased capacity for the production of the key immunoregulatory cytokine, interleukin-2. However, it has not been determined if such changes influence the response of the immune effector cells. Thus, the purpose of the present study was

Julita A Teodorczyk-Injeyan; Marion McGregor; Richard Ruegg; H Stephen Injeyan



Inhibitors of glycoprotein processing alter T-cell proliferative responses to antigen and to interleukin 2.  

PubMed Central

Most of the cell-surface molecules involved in T-cell immune responses are N-linked glycoproteins. We have investigated the effects of inhibitors of glycoprotein processing on specific T-cell functions, with the dual aims of examining the functional role of carbohydrate and of testing the usefulness of such compounds as immunomodulators. Treatment of a cloned murine helper T-cell line with these inhibitors differentially affects the proliferative response of the cell, depending upon the nature of the stimulus. Treatment with the plant alkaloid swainsonine, which inhibits the processing mannosidase II and causes the accumulation of glycoproteins bearing hybrid-type oligosaccharide structures, enhances the proliferative response of the T-cell clone to antigen and to the mitogen concanavalin A. Treatment with another plant alkaloid, castanospermine, which inhibits glucosidase I and causes the accumulation of glucose-containing high-mannose structures, has the opposite effect and inhibits the proliferative response of the T cell to antigen. Cell-surface oligosaccharide alteration does not affect antigen recognition, as judged by the lack of effect of either drug on interleukin 2 production following antigen stimulation. Cells treated with either alkaloid proliferate poorly to exogenous interleukin 2 and may have defective interleukin 2 receptor function. Swainsonine-treated cells apparently have compensatory alterations that can overcome the reduced responsiveness to interleukin 2. Antibody-binding studies indicate that normal quantities of many cell-surface molecules, including the T-cell receptor for antigen, are expressed by the treated cells.

Wall, K A; Pierce, J D; Elbein, A D



Interleukin 2 Acts as an Adjuvant to Increase the Potency of Inactivated Rabies Virus Vaccine  

Microsoft Academic Search

Interleukin 2 (IL-2) occupies a central position in the cascade of events involved in the immune response. We were interested in determining whether IL-2 could function as an adjuvant to vaccination, to increase the immune response to vaccine immunogens. Using the National Institutes of Health test for rabies vaccine potency, we found that daily systemic administration of IL-2 in conjunction

Jack H. Nunberg; Michael V. Doyle; Sonia M. York; Charles J. York



Interleukin2 receptor ? chain regulates the size and content of the peripheral lymphoid compartment  

Microsoft Academic Search

Interleukin-2 receptor ? chain (IL-2R?) expression occurs at specific stages of early T and B lymphocyte development and is induced upon activation of mature lymphocytes. Young mice that lack IL-2R? have phenotypically normal development of T and B cells. However, as adults, these mice develop massive enlargement of peripheral lymphoid organs associated with polyclonal T and B cell expansion, which,

Dennis M. Willerford; Jianzhu Chen; Judith A. Ferry; Laurie Davidson; Averil Ma; Frederick W. Alt



Affinity-Purified Interleukin-2 Induces Proliferation of Large but not Small B Cells.  

National Technical Information Service (NTIS)

Immunoaffinity-purified interleukin 2 (IL2) stimulated proliferation of large but not small B cells. Stimulation was observed even when B cells were cultured at very low cell densities (30,000 per microwell containing 0.2 ml of medium). Addition of small ...

J. J. Mond C. Thompson F. D. Finkelman J. Farrar M. Schaefer



Plasma nitrate plus nitrite changes during continuous intravenous infusion interleukin 2  

Microsoft Academic Search

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

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



A Transglutaminase that Converts Interleukin2 into a Factor Cytotoxic to Oligodendrocytes  

Microsoft Academic Search

Regenerating optic nerves from fish produce a factor that is cytotoxic to oligodendrocytes. The cytotoxic factor is recognized by antibodies to interleukin-2 (IL-2) and has the apparent molecular size of a dimer of IL-2. An enzyme, identified as a nerve transglutaminase, was purified from regenerating optic nerves of fish and was found to catalyze dimerization of human IL-2. The dimerized

Shoshana Eitan; Michal Schwartz



Interaction between human interleukin-2-activated natural killer cells and heat-killed germ tube forms of Candida albicans.  


Human interleukin-2-activated natural killer (LAK) cells are able to recognize and to bind to both live and heat-killed germ tube forms of Candida albicans, establishing a wide and intimate contact as revealed by electron microscopic observations. Following the interaction, LAK cells are activated: an increased expression of some cytokine mRNA (in particular, TNF-alpha, GM-CSF, and IFN-gamma) has been revealed by RT-PCR and perforin secretion has been suggested by immunofluorescence microscopy. Nonetheless, neither morphological damage or growth inhibition of fungal target cells have been detected. Instead, evident signs of cell damage could be noticed in interacting LAK cells. Moreover, the observation by transmission electron microscopy of LAK cell-germ tube conjugates revealed the presence of apoptotic cells. The analysis of LAK cell cytotoxic activity against DAUDI cells showed that the lymphocytic effector underwent a significant reduction in its lytic capability after the interaction with C. albicans. The results obtained in this in vitro study seem to indicate that in such an interaction LAK cells cannot directly inhibit or kill the fungal pathogen by using their lytic machinery but they secrete those cytokines which have stimulatory effects on phagocytic cells. The ultimate results are the programmed death of LAK cells and the enhancement of the fungicidal activity exerted by competent cells. PMID:9637762

Arancia, G; Stringaro, A; Crateri, P; Torosantucci, A; Ramoni, C; Urbani, F; Ausiello, C M; Cassone, A



Cytotoxic Activity of Peripheral Blood Mononuclear Leukocytes, Activated by Interleukin-2/?-Cyclodextrin Nanocomposition against Androgen Receptor-Negative Prostate Cancers.  


Nanocomposition comprised of interleukin-2 in suboptimal noneffective concentration and ?-cyclodextrin was studied in vitro. This preparation as well as interleukin-2 in optimal concentration was shown to increase natural killer activity to K-562 cells and cytotoxicity of activated peripheral blood mononuclear cells (PBMCs) against PC-3 and DU 145 cells. At the same time ?-cyclodextrin or interleukin-2 in equimolar concentrations did not influence the spontaneous killer activity of PBMC. This combination of cyclodextrin + interleukin-2 led to the decrease of interleukin-2 effective concentration by an order. This phenomenon could be explained by cyclodextrins ability to promote the formation of nanoparticles with drugs, which results in enhancing their water solubility and bioavailability. Besides, interleukine-2/?-cyclodextrin nanocomposition as opposed to interleukin-2 alone led to increasing the number of not only lymphocytes, but also macrophages contained in activated PBMC population. Application of low concentration of interleukin-2 allowing for good clinical efficiency may significantly mitigate the side effects of the drug and enable to develop adoption of immunotherapy for patients with androgen-resistant prostate cancer. PMID:22084730

Anisimova, Natalia Yu; Sosnov, Andrey V; Ustyuzhanina, Nadezhda E; Baronzio, Gianfranco; Kiselevsky, Mikhail V



Levels of soluble interleukin-2 receptors are predictive of response in patients treated with interleukin-2 and lymphokine-activated killer cells  

Microsoft Academic Search

Soluble interleukin-2 receptor (sIL-2R) ? (CD25) levels were serially determined in the sera of 20 patients who had undergone\\u000a adoptive immunotherapy with high-dose IL-2 and lymphokine-activated killer (LAK) cells for various types of metastatic solid\\u000a tumors or Hodgkin’s disease. The treatment course consisted of 5 days of high-dose IL-2 priming followed by the collection\\u000a of peripheral blood leukocytes by leukapheresis,

Elisabeth Paietta; David L. Nelson; Janet Andersen; Janice P. Dutcher; Peter H. Wiernik



Irreversible inactivation of interleukin 2 in a pump-based delivery environment.  

PubMed Central

The physical stability of pharmaceutical proteins in delivery environments is a critical determinant of biological potency and treatment efficacy, and yet it is often taken for granted. We studied both the bioactivity and physical stability of interleukin 2 upon delivery via continuous infusion. We found that the biological activity of the delivered protein was dramatically reduced by approximately 90% after a 24-hr infusion program. Only a portion of these losses could be attributed to direct protein deposition on the delivery surfaces. Analysis of delivered protein by size exclusion chromatography gave no indication of insulin-like, surface-induced aggregation phenomena. Examination of the secondary and tertiary structure of both adsorbed and delivered protein via Fourier-transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated that transient surface association of interleukin 2 with the catheter tubing resulted in profound, irreversible structural changes that were responsible for the majority of the biological activity losses.

Tzannis, S T; Hrushesky, W J; Wood, P A; Przybycien, T M



Cloning and chromosomal mapping of bovine interleukin-2 receptor gamma gene.  


Interleukin-2 receptor (IL-2R) gamma chain, a member of the cytokine receptor superfamily, forms a high-affinity receptor with IL-2R alpha and beta chains that plays an important role in interleukin-2 (IL-2) signal transduction. We have cloned and characterized the bovine IL-2Rgamma gene and corresponding cDNA. Bovine IL-2Rgamma is a single-copy gene that contains 8 exons and spans approximately 3.8 kb. The promoter region lacks conventional TATA and CCAAT consensus sites, but contains several regulatory elements that are recognition sites for the GATA binding proteins, AP-1 and AP-2. Physical assignment by fluorescence in situ hybridization (FISH) placed the bovine IL-2Rgamma gene on chromosome Xq23. PMID:8672241

Yoo, J; Stone, R T; Solinas-Toldo, S; Fries, R; Beattie, C W



Improved Immunotherapy of a Recombinant Carcinoembryonic Antigen Vaccinia Vaccine When Given in Combination with Interleukin2  

Microsoft Academic Search

Interleukin-2 (IL-2) has been an effective immune modulator in several active-specific immunotherapy experimental protocols using either viral or oncolysate-based vaccines. In this report, data indicate that IL-2 ad ministration can appreciably augment the therapeutic effect of a single immunization of a recombinant vaccinia virus-carcinoembryonic antigen (rV-CEA) vaccine using a CEA-expressing syngeneic experimental mu rine model system. A single rV-CEA immunization

Joanne P. McLaughlin; Jeffrey Schlom; Judy A. Kantor; John W. Greiner


Soluble Interleukin 2 Receptor and Tissue Polypeptide Antigen Serum Concentrations in End-Stage Renal Failure  

Microsoft Academic Search

Serum concentrations of soluble interleukin 2 receptor (IL-2R) were measured in 65 hemodialysis patients and compared with serum levels of ?2-microglobulin and tissue polypeptide antigen (TPA). Elevated IL-2R levels, found in 85% of examined patients, correlated with elevated TPA serum concentrations (p < 0.05). Patients with high IL-2R levels were significantly younger (p < 0.05) than patients with low levels.

Gerd Walz; Ulrich Kunzendorf; Oliviera Josimovic-Alasevic; Lothar Preuschoff; Anke Schwarz; Frieder Keller; Gernot Asmus; Gerd Offermann; Tibor Diamantstein; Armin Distler




Microsoft Academic Search

Interleukin 2 (IL-2), 1 also known as T cell growth factor, discovered by Morgan et al. (1), is a lymphokine produced by normal peripheral blood lymphocytes after antigen or mitogen stimulation (2, 3) and is required for the proliferation and function of T cells (2, 3), natural killer cells (4-6), and cytotoxic effector cells in vitro and in vivo (7-9).



cDNA cloning of porcine interleukin 2 by polymerase chain reaction.  


Porcine interleukin 2 (IL-2) cDNA was cloned by polymerase chain reaction (PCR), using primers derived from the corresponding bovine sequence. The resulting porcine DNA sequence encodes a 154 residue IL-2 primary translation product. Comparison of the mature, secreted form of porcine IL-2 with those of other species was carried out in an attempt to identify differences that might contribute to the observed differing species specificities. PMID:2054386

Goodall, J C; Emery, D C; Bailey, M; English, L S; Hall, L



Fast Evolution of Interleukin2 in Mammals and Positive Selection in Ruminants  

Microsoft Academic Search

.   Interleukin-2 (IL-2) is a cytokine involved in induction and regulation of the immune response in mammals. There have been\\u000a numerous reports about the search for IL-2 in species other than mammals, and recently an IL-2-like gene has been isolated\\u000a in chicken. Using PCR, we searched for IL-2 gene sequences in a wide variety of mammals, including marsupials and monotremes,

Dominique Zelus; Marc Robinson-Rechavi; Myriam Delacre; Claude Auriault; Vincent Laudet



Effect of Pregnancy and Human Immunodeficiency Virus Infection on Intracellular Interleukin2 Production Patterns  

Microsoft Academic Search

Human immunodeficiency virus type 1 (HIV-1) infection decreases the production of interleukin-2 (IL-2) from CD4 and CD8 T cells. Recombinant IL-2 (rIl-2) has been given to HIV-infected individuals to generate significant increases in CD4 T-cell counts. There are limited data regarding the effects of pregnancy and HIV infection on IL-2 production in humans. To investigate the effects of human pregnancy,

Madeline Y. Sutton; Bart Holland; Thomas N. Denny; Ambrosia Garcia; Zenaida Garcia; Dana Stein; Arlene D. Bardeguez



Ability of isoprinosine to restore interleukin-2 production and T cell proliferation in autoimmune mice.  

PubMed Central

Autoimmune mice bearing the single autosomal recessive gene 1pr are unable to produce the T cell growth factor, interleukin-2 (IL-2). A physiological consequence of this defect is the inability of T cells from C57B1/6J-lpr/lpr mice to respond to antigen presented by macrophages. In an attempt to reverse these abnormalities, we administered the inosine containing drug isoprinosine. Injection of isoprinosine after antigen immunization restored both antigen presentation and IL-2 production.

Fischbach, M; Talal, N



Lymphocyte subset, lymphocyte proliferative response, and soluble interleukin-2 receptor in anorexic patients  

Microsoft Academic Search

Background: Despite a prominent malnourished state, anorexics are unexpectedly free from infection. Several studies have shown that the cell-mediated immunity of anorexics might be well preserved, but results are conflicting.Methods: Lymphocyte subsets, lymphoproliferative response to phytohemagglutinin, and soluble interleukin-2 receptor (sIL-2R) were measured in 7 patients with anorexia nervosa restricting type (RAN), 6 with anorexia nervosa binge-eating\\/purging type (ANBP), and

Toshihiko Nagata; Nobuo Kiriike; Wataru Tobitani; Youjirou Kawarada; Hisato Matsunaga; Sakae Yamagami



Determination of capillary leakage due to recombinant interleukin-2 by means of noninvasive conductivity measurements  

Microsoft Academic Search

Summary  One of the most common side effects of treatment with recombinant interleukin-2 (IL-2) is capillary leakage. Its genesis is not completely understood. The aim of the study was to determine whether capillary leakage can be monitored by means of a non-invasive conductivity technique and to study its starting point. Eight patients with advanced renal cell cancer were studied in a

Cornelis G. Olthof; Johanna W. Baars; John Wagstaff; Ab J. M. Donker; Hans Schneider; Peter M. J. M. de Vries



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

Microsoft Academic Search

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

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



Inhibition of Stimulated Interleukin2 Production in Whole Blood: A Practical Measure of Cyclosporine Effect  

Microsoft Academic Search

Background: Prediction of cyclosporine (CSA) efficacy and toxicity in individual patients is difficult. There is no practical, biologically relevant, pharmacodynamic measure of CSA effect. A major effect of CSA is to decrease interleukin-2 (IL-2) production; however, mea- surement of this effect in isolated lymphocytes as a marker of response to CSA has been problematic. Methods: CSA inhibition of phytohemagglutinin-P (PHA)-stimulated

C. Michael Stein; John J. Murray; Alastair J. J. Wood


Differential Regulation of Colony-Stimulating Factors and Interleukin 2 Production by Cyclosporin A  

Microsoft Academic Search

Stimulation of T lymphocytes with mitogens or antigens is followed by proliferation and lymphokine production. Although cyclosporin A (CsA), an immunosuppressive drug, has been shown to inhibit the production of certain lymphokines, including interleukin 2 (IL-2), interleukin 3 (IL-3), and gamma -interferon, its effect on the production of granulocyte\\/macrophage colony-stimulating factor (GM-CSF) has not been evaluated. In the current study,

M. Bickel; H. Tsuda; P. Amstad; V. Evequoz; S. E. Mergenhagen; S. M. Wahl; D. H. Pluznik



Phase II Trial of Systemic Recombinant Interleukin2 in the Treatment of Refractory Nasopharyngeal Carcinoma  

Microsoft Academic Search

Background: Interleukin-2 (IL-2) is a cytokine produced by activated T cells, which has shown powerful immunostimulatory and antineoplastic properties. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated cancer with abundant lymphocyte infiltration histologically. The activity of IL-2 in the treatment of NPC patients is currently unknown. A phase II study was, therefore, initiated to evaluate the efficacy, toxicity and immunological consequences

Kwan-Hwa Chi; Jeffrey N. Myers; Kuan C. Chow; Wing K. Chan; Yuk-Wah Tsang; Yee Chao; Sang H. Yen; Michael T. Lotze



Phase I Evaluation of Combination Therapy with Interleukin 2 and 7Interferon1  

Microsoft Academic Search

Kecombinant interleukin 2 (II.-2) is a potent inducer of lymphokine- activated killer (I.AK) activity directed against autologous and allogeneic tumors; these effects are mediated by CD3-ncgative, CD56-positive, and CI)16-positive lymphocytes. Although IL-2 therapy has been associated with clinical responses, particularly in patients with renal cell carcinoma and melanoma, these responses have occurred with high, toxic doses of this cytokine. Since

Louis M. Weiner; Kristin Padavic-Shaller; Joanne Kitson; Perry Watts; Robert L. Krigel; Samuel Litwin


Regression of Bladder Tumors in Mice Treated with Interleukin 2 Gene-Modified Tumor Cells  

Microsoft Academic Search

Summary This study explored the use of interleukin 2 (I1,2) and interferon ~\\/(IFN-3') gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner

John Connor; Rajat Bannerji; Shiro Saito; Warren Heston; William Fair; Eli Gilboa


Interleukin 2 Receptors and Detergent-Resistant Membrane Domains Define a Clathrin-Independent Endocytic Pathway  

Microsoft Academic Search

Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes

Christophe Lamaze; Annick Dujeancourt; Takeshi Baba; Charles G. Lo; Alexandre Benmerah; Alice Dautry-Varsat



The role of interleukin-2 during homeostasis and activation of the immune system  

Microsoft Academic Search

Interleukin-2 (IL-2) signals influence various lymphocyte subsets during differentiation, immune responses and homeostasis. As discussed in this Review, stimulation with IL-2 is crucial for the maintenance of regulatory T (TReg) cells and for the differentiation of CD4+ T cells into defined effector T cell subsets following antigen-mediated activation. For CD8+ T cells, IL-2 signals optimize both effector T cell generation

Jonathan Sprent; Onur Boyman



Alteration of dacarbazine pharmacokinetics after interleukin-2 administration in melanoma patients  

Microsoft Academic Search

In an effort to improve the treatment of metastatic malignant melanoma, we evaluated the sequential administration of the chemotherapeutic agent dacarbazine (DTIC) and the biological response modifier interleukin-2 (rIL-2) in a phase I–II study. Since the combination of biological response modifiers and chemotherapeutic agents could alter drug disposition, we evaluated the pharmacokinetics of DTIC and its major metabolite, 5-aminoimidazole 4-carboxamide

Guy G. Chabot; Lawrence E. Flaherty; Manuel Valdivieso; Laurence H. Baker



Phosphatidylinositol 3Kinase Couples the Interleukin2 Receptor to the Cell Cycle Regulator E2F  

Microsoft Academic Search

Cell cycle progression initiated by interleukin-2 (IL-2) in T cells is critical for lymphoproliferation and an immune response. Phosphatidyl inositol 3-kinase (PI3K) is activated by IL-2. However, nuclear targets for PI3K are not known. Here we identify the cell cycle regulator E2F as an IL-2 target in T lymphocytes and PI3K as the critical signaling pathway. We eliminate both Stat5

Paul Brennan; Jane W Babbage; Boudewijn M. T Burgering; Bernd Groner; Karin Reif; Doreen A Cantrell



Serum soluble interleukin-2 receptor: A useful indicator of the clinical course in pulmonary tuberculosis  

Microsoft Academic Search

Setting: In tuberculosis both host protection and most pathogenic mechanisms depend on T lymphocytes. After activation by mycobacterial antigens, T cells both secrete interleukin-2 (IL-2) and express a high affinity receptor for this molecule (IL-2R) on their own surface. A soluble fraction of IL-2 receptor (sIL-2R), released from cell membrane, is detectable in serum and its concentration is known to

M. J. Avilés Inglés; C. Contessotto; J. Ontañón Rodriguez; A. García Alonso; M. Muro Amador; M. Canteras Jordana; F. Sánchez Gascón; R. Alvarez López



Antiproliferative effect of polyunsaturated fatty acids and interleukin-2 on normal and abnormal human lymphocytes  

Microsoft Academic Search

The polyunsaturated fatty acids (PUFAs), linoleic acid (LA), alpha linolenic acid (ALA), gamma linolenic acid (GLA), arachidonic acid (AA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), showed inhibition of growth of both normal and abnormal (Molt-4) human lymphocytes, and inhibition was concentration-dependent. Interestingly, the production of the lymphokine Interleukin-2 (IL-2) was elevated in Molt-4 cells, but it was reduced in

M. Ambika Devi; N. P. Das



The interleukin-2 receptor in lesions and serum of bullous pemphigoid  

Microsoft Academic Search

The interleukin-2 receptor (IL-2R) is mainly expressed on activated T cells. Depending on its rate of synthesis, a portion is released from the cell surface as soluble IL-2R (sIL-2R). Since the role of mononuclear cells in the pathology of bullous pemphigoid (BP) is not well understood, we determined the sIL-2R in both blister fluid and serum of 15 BP patients

D. Zillikens; A. Ambach; M. Schuessler; R. Dummer; A. A. Hartmann; G. Burg



Effects of Sleep and Sleep Deprivation on Catecholamine And Interleukin2 Levels in Humans: Clinical Implications  

Microsoft Academic Search

The objective of this study was to evaluate the effects of nocturnal sleep, partial night sleep deprivation, and sleep stages on catechol- amine and interleukin-2 (IL-2) levels in humans. Circulating levels of catecholamines and IL-2 were sampled every 30 min during 2 nights: undisturbed, baseline sleep and partial sleep deprivation-late night (PSD-L; awake from 0300 - 0600 h) in 17




Effect of 1,1-dimethylhydrazine on lymphoproliferation and interleukin 2 immunoregulatory function  

Microsoft Academic Search

The studies reported here suggest that the immunomodulatory effects of 1,1-dimethylhydrazine (UDMH) are associated, in part, with interference with interleukin 2 (IL-2) regulatory action. Concanavalin A (Con A)-stimulated DNA synthesis in murine splenocytes was inhibited from 18.6 to 44.1% at sub-toxic concentrations of UDMH (10 to 50 µg\\/ml) and IL-2-dependent DNA synthesis in CTLL-20 cells was inhibited from 11.3 to

Richard M. Bauer; Melinda J. Tarr; Richard G. Olsen



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

Microsoft Academic Search

Sixty-six patients with disseminated malignancy were treated with recombinant interleukin-2 (IL-2) on a three times a week (M, W, F) IV-bolus injection schedule. Doses ranged from 0.001 to 14.0 × 106 units\\/M2 body surface area. Consecutive groups of 3-5 patients were placed on each dose level and were maintained on that level except for dosage de-escalation for toxicity. Toxicity to

Evan M. Hersh; J. Lee Murray; Waun Ki Hong; Michael G. Rosenblum; James M. Reuben; Robert Weilbaecher; Amar N. Sarwal; Edward C. Bradley; Michael Konrad; Frank C. Arnett



Differential effects of ether lipids on the activity and secretion of interleukin-1 and interleukin-2  

Microsoft Academic Search

Alkyl lysophospholipids (ALP) are synthetic analogues of lysophosphatidylcholine and represent a new generation of antitumor\\u000a drugs currently being tested in phase-I trials in patients with cancer. The present study reports the differential modulation\\u000a of human immunocompetent cells in vitro by ALP. Serum-bound ALP effectively blocked the response of growth factor-dependent\\u000a cells to interleukin-2 (IL-2), inhibited the cellular production and release

Reinhard Andreesen; Veronika Giese



Effects of Recombinant Interleukin-1 and Interleukin-2 on Human Keratinocytes Running Head: Interleukins-1 and 2 Effect on Keratinocytes,  

National Technical Information Service (NTIS)

The effects of recombinant interleukin-1 alpha and beta, as well as recombinant interleukin-2, on human keratinocyte proliferation were studied in serum-containing as well as defined media. Both interleukin-1 preparations did not stimulate keratinocyte gr...

A. B. Cua G. J. Wastek J. N. Mansbridge V. B. Morhenn



Bovine T lymphocytes. I. Generation and maintenance of an interleukin-2-dependent, cytotoxic T-lymphocyte cell line.  

PubMed Central

Primary and secondary bovine allogeneic mixed leucocyte cultures were examined for the generation of antigen-specific cytotoxic leucocytes. While optimal generation of murine and human cytotoxic T lymphocytes typically requires 4-8 days, alloantigen-specific cytotoxic bovine leucocytes were demonstrated consistently only after prolonged incubation periods, optimally found to be about 15 days. Restimulation of long-term bovine mixed leucocyte cultures with the original stimulator population revealed responder cells demonstrating augmented alloantigen-specific lytic activity. When placed into human recombinant interleukin-2, responder cells expanded and required passaging every 3-4 days. The same was not true of cells placed into interleukin-2-free medium. Cells cultured in interleukin-2-containing medium retained alloantigen specificity after 10 weeks of culture. Moreover, they continued to display total dependence on human, simian or bovine interleukin-2 for growth.

Picha, K S; Baker, P E



Studies Evaluating the Antitumor Activity and Toxicity of Interleukin15, a New T Cell Growth Factor: Comparison with Interleukin2  

Microsoft Academic Search

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target

William Munger; Susan Quinn Dejoy; R. Jeyaseelan; Lawrence W. Torley; Kenneth H. Grabstein; June Eisenmann; Ray Paxton; Tom Cox; Michael M. Wick; S. S. Kerwar



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

NASA Astrophysics Data System (ADS)

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.

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



Appraisal of rabies vaccine potency by determination of in vitro, specific interleukin-2 production.  


The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed by the production of interleukin-2 (IL-2) by CD4+CD8- lymphocytes. IL-2 production by splenocytes from mice immunized with various vaccines was measured following in vitro stimulation with antigens from different rabies and rabies-related strains. IL-2 production was specific, reproducible and correlated with the vaccine protective activity as determined by the pre-exposure NIH test. Our results suggest that measurement of IL-2 production could be used for the appraisal of rabies vaccine potency. PMID:1888490

Joffret, M L; Zanetti, C; Morgeaux, S; Leclerc, C; Sureau, P; Perrin, P



Plasma Soluble Interleukin2 Receptor (sIL2R) Levels in Patients with Acute Leukemia  

Microsoft Academic Search

The plasma soluble interleukin-2 receptor (sIL-2R) level was higher in 137 patients with acute leukemia (1,489 ± 1,798U\\/ml, including 98 cases of acute myeloid leukemia (AML), 1,063 ± 1,414 U\\/ml, and 39 cases of acute lymphoblastic leukemia (ALL), 2,561 ± 2,194 U\\/ml), compared to 49 normal control subjects, 421±151 U\\/ml). The ALL patients showed elevated plasma sIL-2R levels more frequently

Yeonsook Moon; Yonggoo Kim; Myungshin Kim; Jihyang Lim; Chang Suk Kang; Won Il Kim; Sang In Shim; Nak Gyun Chung; Yoon Hee Park; Woo Sung Min; Kyungja Han


Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis.  

PubMed Central

We have analyzed structure--function relationships of the protein hormone murine interleukin 2 by fine structural deletion mapping. A total of 130 deletion mutant proteins, together with some substitution and insertion mutant proteins, was expressed in Escherichia coli and analyzed for their ability to sustain the proliferation of a cloned murine T cell line. This analysis has permitted a functional map of the protein to be drawn and classifies five segments of the protein, which together contain 48% of the sequence, as unessential to the biological activity of the protein. A further 26% of the protein is classified as important, but not crucial, for the activity. Three regions, consisting of amino acids 32-35, 66-77 and 119-141 contain the remaining 26% of the protein and are critical to the biological activity of the protein. The functional map is discussed in the context of the possible role of the identified critical regions in the structure of the hormone and its binding to the interleukin 2 receptor complex. Images

Zurawski, S M; Zurawski, G



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


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

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



Thyroiditis after treatment with interleukin-2 and interferon alpha-2a.  

PubMed Central

Serial thyroid functions studies were carried out in patients with melanoma and renal cell carcinoma treated with interleukin-2 (3 MU m-2 by continuous infusion days 1-4) and interferon alpha-2a (6 MU m-2 subcutaneously on days 1 and 4), both given on alternate weeks. The results on eight patients who completed at least three cycles of treatment are described. Four patients developed thyroid dysfunction with a hyperthyroid phase of 2 weeks followed by a hypothyroid phase ranging from 12 to 24 weeks. Two patients became clinically symptomatic and required treatment. Fine-needle aspirates of the thyroid were obtained in three patients with thyroid dysfunction. The cytology revealed a mixed cellular infiltrate with lymphocytes and histiocytes, and immunocytochemical staining showed strong HLA-DR expression of all thyrocytes, both suggestive of an autoimmune thyroiditis. One patient with thyroiditis developed anti-thyroglobulin antibodies, the serology of all other patients was normal. Patients with thyroid dysfunction tended to have higher in vivo stimulated lytic activity of peripheral mononuclear blood cells and had significantly higher levels of CD16 positive blood cells as compared to euthyroid patients. The possibility of autoimmune thyroiditis should be anticipated in future trails combining interleukin-2 and interferon alpha-2a. Images Figure 2 Figure 3

Pichert, G.; Jost, L. M.; Zobeli, L.; Odermatt, B.; Pedia, G.; Stahel, R. A.



Demonstration of a non-Tac peptide that binds interleukin 2: a potential participant in a multichain interleukin 2 receptor complex.  

PubMed Central

The interleukin 2 (IL-2) receptor system plays a key role in the T-cell immune response. Although IL-2 binding was reported to be restricted to the Tac peptide, we have identified an IL-2 binding peptide that does not react with anti-human IL-2 receptor monoclonal antibodies, including anti-Tac on MLA 144, a gibbon ape T-cell line. The MLA 144 cell line expressed 6800 IL-2 binding sites per cell with a low (Kd = 14 nM) affinity for human recombinant IL-2. Using cross-linking methodology, we demonstrated that the IL-2 binding peptide on MLA 144 is larger (Mr 75,000) than the Tac peptide, which has a Mr of 55,000. An IL-2 binding peptide of similar size (Mr 75,000) was also identified in addition to the Tac peptide (Mr 54,000-57,000) on Hut 102, a human T-cell lymphotrophic virus I-induced T-cell leukemia line, and phytohemagglutinin-activated normal human and gibbon ape lymphoblasts. Anti-Tac antibody did not block the binding of 125I-labeled IL-2 to MLA 144 cells. However, this antibody abolished the binding of 125I-labeled IL-2 not only to the Tac peptide on Hut 102 cells and normal lymphoblasts but also to the Mr 75,000 IL-2 binding peptide, suggesting that this latter peptide is associated with the Tac peptide to form the high-affinity IL-2 receptor complex. Images

Tsudo, M; Kozak, R W; Goldman, C K; Waldmann, T A



Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa.  


To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa-inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10-35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression of CD4+ and CD8+ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4+ and CD8+ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4+ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8+ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals (P < 0.0001), coinciding with an increase in CD8+ T cells numbers in these cultures. The data show that factors besides IL-2; and probably an imbalance in the percentages of CD4+ and CD8+ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa-inoculated rhesus monkeys. PMID:9278942

Dennis, V A; Osae-Addo, G; Lowrie, R C



T cells, but not B cells, are required for bowel inflammation in interleukin 2-deficient mice  

PubMed Central

Interleukin-2 (IL-2)-deficient (IL-2-/-) mice develop anemia and colonic inflammatory bowel disease. To elucidate the mechanism of this disease, we have bred IL-2-/- mice to two strains of immunodeficient mice, RAG-2-deficient (RAG-2-/-, lacking B and T cells) and JH- deficient mice (JH-/-, lacking B cells). IL-2-/-, RAG-2-/- double- mutant mice are disease free, while IL-2-/-, JH-/- double-mutant mice succumb to bowel disease at the same rate as IL-2-/- littermates. IL-2- /-, JH-/- mice do not, however, succumb to anemia. Thus, spontaneous intestinal inflammation in IL-2-/- mice requires mature T cells, not B cells, while anemia is dependent on B cells.



Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library.  


Libraries of chemical compounds individually coupled to encoding DNA tags (DNA-encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high-quality DNA-encoded chemical library comprising 30,000 drug-like compounds; this was screened in 170 different affinity capture experiments. High-throughput sequencing allowed the evaluation of 120?million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor-associated antigen carbonic anhydrase?IX (CA?IX) and the pro-inflammatory cytokine interleukin-2 (IL-2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL-2 was confirmed by molecular docking. Our findings suggest that DNA-encoded chemical libraries allow the facile identification of drug-like ligands principally to any protein of choice, including molecules capable of disrupting high-affinity protein-protein interactions. PMID:22588840

Leimbacher, Markus; Zhang, Yixin; Mannocci, Luca; Stravs, Michael; Geppert, Tim; Scheuermann, Jörg; Schneider, Gisbert; Neri, Dario



Augmentation of murine hematopoiesis by interleukin 2-activated irradiated T cells.  


We have examined the role of T cells activated with interleukin-2 (IL-2) in vitro and subsequently irradiated (2500 rads), in stimulating murine hematopoiesis in a syngeneic system. Our data suggest that activated, irradiated T (AIT) cells significantly increased the progenitor cell activity of T cell-depleted bone marrow (BM) both in vitro and in vivo as compared with controls (P < 0.001). The efficacy of AIT cells was comparable to that of activated, nonirradiated T (AT) cells (P > 0.05). Optimal stimulation of BM progenitor cell activity was seen when T cells were activated for 4 days and used in a BM to T cell ratio of 1:2 or 1:5. The effect of these activated cells was related to the release of factors with ability to enhance hematopoiesis. These observations may have implications in enhancing the engraftment of T cell-depleted BM in allogeneic transplantation. PMID:7570956

Charak, B S; Brown, E G; Mazumder, A



Interleukin-2 inhibits the GABA-induced Cl- current in identified Aplysia neurons.  


The effects of extracellularly applied recombinant human interleukin-2 (rhIL-2) on the gamma-aminobutyric acid (GABA)-induced Cl- current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied rhIL-2 (10-40 U/ml) reduced the GABA-induced current in the neurons without affecting resting membrane conductance and the holding current. The suppressing effect of rhIL-2 on the current was completely reversible. Heat-inactivated rhIL-2 was without effect. These results suggest that the immunomodulator IL-2 can modulate the GABA-induced response in the nervous system. PMID:1281891

Sawada, M; Hara, N; Ichinose, M



B-Lymphocyte Proliferation during Bovine Leukemia VirusInduced Persistent Lymphocytosis Is Enhanced by T-Lymphocyte-Derived Interleukin-2  

PubMed Central

Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5+ B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-?) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.

Trueblood, Esther S.; Brown, Wendy C.; Palmer, Guy H.; Davis, William C.; Stone, Diana M.; McElwain, Terry F.



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

Microsoft Academic Search

Recombinant human interleukin-2 (rhIL-2) therapy is approved for treating patients with advanced melanoma yet significant\\u000a responses are observed in only 10–15% of patients. Interleukin-2 induces Foxp3 expression in activated human CD8 T cells in\\u000a vitro and expands circulating CD8 Foxp3+ T cells in melanoma patients. Employing IL-2 responsive (B16-F1, B16-BL6, JB\\/MS,\\u000a MCA-205) and nonresponsive (JB\\/RH, B16-F10) subcutaneous tumor mouse models,

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


In Vivo Induction of the Lymphokine-activated Killer Phenomenon:Interleukin 2- dependent Human Non-Major Histocompatibility Complex-restricted Cytotoxicity Generated in Vivoduring Administration of Human Recombinant Interleukin 2  

Microsoft Academic Search

The availability of purified human recombinant interleukin 2 (IL-2) has enabled clinical trials to test its in vivo effects. We report here the imntunological effects of 7 consecutive days of IL-2 treatment given to 25 patients with cancer in a clinical Phase I study. Peripheral blood lymphocytes obtained from patients following therapy with IL-2 had enhanced proliferative responses to IL-2

Jacquelyn A. Hank; Peter C. Kohler; Gilda Weil-Hillman; Nancy Rosenthal; Karen H. Moore; Barry Storer; Debbie Minkoff; Jeff Bradshaw; Robin Bechhofer; Paul M. Sondel


Simulated microgravity inhibits the genetic expression of interleukin-2 and its receptor in mitogen-activated T lymphocytes  

Microsoft Academic Search

Experiments conducted in space in the last two decades have shown that T lymphocyte activation in vitro is remarkably reduced in microgravity. The data indicate that a failure of the expression of the interleukin-2 receptor (measured as protein secreted in the supernatant) is responsible of the loss of activity. To test such hypothesis we have studied the genetic expression of

Isabelle Walther; Proto Pippia; Maria Antonia Meloni; Franco Turrini; Franca Mannu; Augusto Cogoli



Hematologic and Immunologic Effects of the Systemic Administration of Recombinant Interleukin2 After Autologous Bone Marrow Transplantation  

Microsoft Academic Search

T cells from allogeneic bone marrow grafts are responsible for a graft versus leukemia effect. Use of recombinant Interleukin-2 (rlL-2) after autologous bone marrow trans- plantation (BMT) may enhance immune function and hope- fully reproduce the allogeneic reaction. We report here the hematologic and immunologic changes observed in the first 10 patients of a phase 1 trial studying the infusion

D. Blaise; D. Olive; A. M. Stoppa; P. Wens; C. Pourreau; M. Lopez; M. Attal; C. Jasmin; G. Monges; C. Mawas; P. Mannoni; P. Palmer; C. Franks; T. Philip; D. Maraninchi



Enhancement of Antibody-Dependent Cellular Cytotoxicity of Neonatal Cells by Interleukin2 (IL2) and IL12  

Microsoft Academic Search

Newborn infants are more susceptible to infections due in part to deficiencies in the cytotoxic functions of their lymphocytes. We investigated the ability of interleukin-2 (IL-2) and IL-12 to enhance the cytotoxicity of neonatal (cord blood) and adult mononuclear cells (MNCs) in both natural killer (NK) cell and antibody- dependent cellular cytotoxicity (ADCC) assays. The cytotoxic activity of cord blood




A systematic review of the relation between interleukin-2 schedule and outcome in patients with metastatic renal cell cancer  

Microsoft Academic Search

In Europe, interleukin 2 (IL-2) is one of the two treatment modalities officially approved for patients with metastatic renal cell cancer. Traditionally, IL-2 has been administered by three different routes: intermittent bolus injection (BIV), continuous intravenous infusion (CIV) and subcutaneous injection (SC). There have been few randomized trials designed to compare these routes of administration. This paper describes a systematic

G Baaten; A. C Voogd; J Wagstaff



Induction of Graft versus Leukemia Effect in Bone Marrow Transplantation: Dosage and Time Schedule Dependencyof Interleukin 2 Therapy1  

Microsoft Academic Search

The present work is a continuation of our studies to improve the graft versusleukemia (GVL) effect in autologousbone marrowtransplantation. We have recently shown that the GVL effect of bone marrow transplan tation (BMT) with interleukin 2 (IL-2)-activated bone marrow (ABM) followed by IL-2 therapy immediately after BMT is superior to the GVL effect of BMT with fresh, syngeneic bone marrow,

Bishan S. Charak; Russell K. Brynes; Suzy Katsuda; Susan Groshen; Su-Chiu Chen; Amitabha Mazumder



Deficient interleukin 2 production in rheumatoid arthritis: association with active disease and systemic complications.  

PubMed Central

Interleukin 2 (IL-2) production and proliferative responses of peripheral blood mononuclear cells (PBMC) stimulated with three concentrations of PHA were measured in 75 patients with rheumatoid arthritis (RA) and 25 normal controls. All patients were on a standard therapeutic regime, and were assessed for disease activity by clinical and laboratory criteria. Rheumatoid cells showed significantly lower IL-2 production and proliferation than normal PBMC at all PHA doses. These differences were not attributable to different kinetics. Within the rheumatoid population, both IL-2 levels and proliferation were lower in patients with active disease than those with inactive RA. Patients with extra-articular disease showed the most pronounced defects. Proliferative responses showed an inverse correlation with clinical indices of disease activity but not with measures of the acute phase response. Rheumatoid patients had higher proportions of CD4+, TFR+ and Tac+ lymphocytes than controls. Both proliferative responses and IL2- levels showed a positive relationship with the proportion of CD4+ cells, and an inverse relationship with Tac+ lymphocytes. Monocyte depletion and partial reconstitution resulted in an increase of both proliferation and IL-2 production, which was more marked in RA patients, suggesting that depressed IL-2 production may relate in part to monocyte effects. However, this cannot completely explain the magnitude of the defects observed, because normal monocytes did not increase the responses of rheumatoid lymphocytes, neither did rheumatoid monocytes suppress the responses of normal lymphocytes.

Kitas, G D; Salmon, M; Farr, M; Gaston, J S; Bacon, P A



Soluble interleukin-2 receptor, intercellular adhesion molecule-1 and interleukin-10 serum levels in patients withelanoma  

PubMed Central

Serum soluble interleukin-2 receptor (sIL-2R), intercellular adhesion molecule-1 (sICAM-1) and interleukin-10 (IL-10) have each been reported as useful markers for melanoma progression. To evaluate the clinical relevance of these three markers, we simultaneously analysed their serum levels in patients with melanoma. A longitudinal study with a 3-year follow-up was performed and different stages of the disease were considered. Mean values of sIL-2R were significantly higher than in normal controls in all stages and correlated with the disease progression. The prognosis of patients with levels > 529 U/ml of sIL-2R was significantly poorer than in patients with sIL-2R levels < 529 U/ml. Levels of sICAM-1 were also elevated in melanoma patients, specially at the time of the metastatic disease. Serum IL-10 levels were more frequently detectable in the patients that developed metastasis during follow-up, and the prognosis of patients with detectable IL-10 levels was significantly poorer than in those patients with IL-10 undetected levels. Statistical analysis based on Logistic and Cox regression models showed that only sex, stage and sIL-2R value are factors significantly associated with metastatic progression. Moreover, high levels of sIL-2R could be a risk factor for malignant progression in melanoma. © 2000 Cancer Research Campaign

Boyano, M D; Garcia-Vazquez, M D; Lopez-Michelena, T; Gardeazabal, J; Bilbao, J; Canavate, M L; Galdeano, A Garcia De; Izu, R; Diaz-Ramon, L; Raton, J A; Diaz-Perez, J L



Retinoic acid inhibits in vivo interleukin-2 gene expression and T-cell activation in mice  

PubMed Central

Interleukin-2 (IL-2) is an essential cytokine for T-lymphocyte homeostasis. We have previously reported that all-trans retinoic acid (atRA) enhances the secretion of IL-2 from human peripheral blood T cells in vitro, followed by increased proliferation and inhibition of spontaneous cell death. In this study we used a transgenic IL-2 gene luciferase reporter model to examine the effects of atRA in vivo. In contrast to the observations in human T cells, we found an overall reduction in luciferase-reported IL-2 gene expression in mice treated with atRA. Whole-body luminescence of anti-CD3-treated and non-treated mice was reduced in mice receiving atRA. Accordingly, after 7 hr, IL-2 gene expression was on average 55% lower in the atRA-treated mice compared with the control mice. Furthermore, mice fed a vitamin A-deficient diet had a significantly higher basal level of luciferase activity compared with control mice, demonstrating that vitamin A modulates IL-2 gene expression in vivo. Importantly, the atRA-mediated inhibition of IL-2 gene expression was accompanied by decreased DNA synthesis in murine T cells, suggesting a physiological relevance of the reduced IL-2 gene expression observed in transgenic reporter mice.

Ertesvag, Aase; Austenaa, Liv M I; Carlsen, Harald; Blomhoff, Rune; Blomhoff, Heidi Kiil



Mast cell interleukin-2 production contributes to suppression of chronic allergic dermatitis  

PubMed Central

SUMMARY The incidence of chronic allergic dermatitis is rapidly increasing. Regulatory control of this disease has not been adequately explored. Here we report that mast cell derived interleukin-2 (IL-2) contributes to the suppression of chronic allergic dermatitis. Mice deficient in IL-2 production, or deficient in mast cells (KitW-sh/W-sh), showed exacerbated dermatitis upon repeated oxazolone challenge when compared to their wild-type counterparts. Adoptive transfer of wild type, but not Il2?/?, mast cells into KitW-sh/W-sh mice dampened the inflammatory response. During the course of disease mast cell expansion occurred at the site of inflammation and also in the spleen, where production of IL-2 by mast cells was markedly enhanced. In the absence of mast cell IL-2 production, the ratio of activated to regulatory T cells at the site of inflammation was increased. Thus, MC-derived IL-2 contributes to the maintenance of suppression in chronic allergic skin inflammation.

Hershko, Alon Y; Suzuki, Ryo; Charles, Nicolas; Alvarez-Errico, Damiana; Sargent, Jennifer L.; Laurence, Arian; Rivera, Juan



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

PubMed Central

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

McKnight, A J; Classon, B J



Attenuation of interleukin 2-induced pulmonary vascular leak syndrome by low doses of oral methotrexate.  


Pulmonary vascular leak induced in mice by interleukin 2 (IL-2) was attenuated by pretreatment with single or multiple doses of oral methotrexate. Methotrexate also attenuated pulmonary vascular leak when either larger doses of IL-2 or when lymphokine-activated killer (LAK) cells or LAK cells plus IL-2 were administered. Lymphoid infiltrates in the lungs of mice treated with IL-2 and methotrexate were significantly lower. The number of mice surviving treatment with high doses of IL-2 was also significantly increased when these mice were treated with methotrexate. Methotrexate prevented the IL-2-induced increase in the number of splenocytes that were asialo GM1+ but had no effect on Lyt 2+ or L3T4+ cell content. A marginal but significant inhibition in the generation of effector splenocytes that were cytolytic to either YAC or MCA-205 tumor targets was observed in mice treated with methotrexate and IL-2. In vivo studies indicated that methotrexate did not compromise the anti-tumor efficacy of treatment regimens that contained IL-2, LAK cells, or IL-2 and LAK cells. These results demonstrate the potential clinical utility of methotrexate in attenuating pulmonary vascular leak induced by IL-2 without compromising its efficacy. One potential mechanism of action of methotrexate is related to its ability to stimulate the release of adenosine followed by the inhibition of the adhesion of leukocytes to the IL-2-activated endothelium. PMID:7585532

DeJoy, S Q; Jeyaseelan, R; Torley, L W; Schow, S R; Wick, M M; Kerwar, S S



Expression of recombinant porcine interleukin-2 and application of its antibody to immunoassays.  


Interleukin-2 plays an important role in T lymphocyte proliferation and immune response regulations. In this study, porcine IL-2 cDNA was cloned from peripheral blood mononuclear cells, and recombinant porcine IL-2 (rpIL-2) was expressed in Escherichia coli. The size of rpIL-2 without signal peptides was about 15 kDa when determined by SDS-PAGE and Western blotting analysis. Anti-rpIL-2 antibody was produced from mice immunized with the purified rpIL-2, and its specificity was examined by Western blotting and ELISA. In the Western blotting assay, anti-rpIL-2 and anti-recombinant human IL-2 (rhIL-2) antibodies specifically recognized rpIL-2 and rhIL-2, respectively. However, anti-rpIL-2 antibody did not recognize rhIL-2, and anti-rhIL-2 antibody also did not react with rpIL-2 in the same assay. In ELISA, anti-rpIL-2 antibody strongly interacted with both rpIL-2 and rhIL-2, and anti-rhIL-2 antibody also efficiently recognized both proteins. Taken together, the specificity of anti-rpIL-2 antibody for rpIL-2 was demonstrated by Western blotting and ELISA. It was also shown that ELISA is more efficient than Western blotting in determining the species cross-reactivity of anti-rpIL-2 antibody. PMID:12514333

Choi, In-Soo; Yoo, Han-Sang



Cloning and chromosomal assignment of the porcine interleukin-2 receptor alpha (IL-2Ralpha) gene.  


Porcine genomic DNA encoding a 55 kDa subunit of interleukin-2 receptor (IL-2R), which is termed alpha chain (IL-2Ralpha), was cloned by repeated plaque hybridization using IL-2Ralpha cDNA as a probe. Two different lambda phage clones, one of which encoded exon 1 and the 5'-upstream flanking region of IL-2Ralpha gene and another encoded the sequence from exon 2 to exon 8, were isolated. By analysis of the 5'-upstream region of the gene, putative binding motifs for transcription factors such as GATA family proteins, Ikaros, NF-kappaB, NF-IL2Ralpha and SRF, were found as described in human, murine and bovine genes. Two additional motifs for STAT4 binding were also found in this region. Moreover, using the FISH technique, we assigned the porcine IL-2Ralpha locus to the distal end of the long arm of chromosome 10 (10q6-qter) where the vimentin gene had been assigned nearby. PMID:10993181

Kokuho, T; Hiraiwa, H; Yasue, H; Watanabe, S; Yokomizo, Y; Inumaru, S



Elevated serum-soluble interleukin-2 receptor levels in patients with anaplastic large cell lymphoma.  


Levels of serum soluble interleukin 2 receptor (sIL-2R) provide a reliable marker of disease activity in patients with hairy cell leukemia and adult T-cell leukemia/lymphoma. The malignant cells in patients with anaplastic large cell lymphoma (ALCL) express CD30 and are usually positive for expression of CD25. We measured serum sIL-2R and soluble CD30 (sCD30) levels in patients with ALCL treated with EPOCH (etoposide, prednisone, Oncovin, Cytoxan, hydroxydaunorubicin) infusional chemotherapy. Serum sCD30 levels were elevated and decreased in response to therapy as previously reported. Serum sIL-2R levels were elevated in 7 of 9 patients with ALCL and decreased in response to treatment. Baseline serum sIL-2R levels varied but correlated well with serum sCD30 levels (r = 0.97). Patients positive for the anaplastic lymphoma kinase (ALK) gene showed elevated sIL-2R levels, whereas those negative for ALK had normal serum sIL-2R levels and their tumors lacked CD25 expression. Serum sIL-2R levels were elevated in both patients with recurrent disease. PMID:15205267

Janik, John E; Morris, John C; Pittaluga, Stefania; McDonald, Kristin; Raffeld, Mark; Jaffe, Elaine S; Grant, Nicole; Gutierrez, Martin; Waldmann, Thomas A; Wilson, Wyndham H



Agonistic effects of a monoclonal antibody specific for the interleukin-2 receptor  

SciTech Connect

Interleukin-2 (IL-2) mediated immune responses can be blocked by monoclonal antibodies to the IL-2 receptor. The monoclonal antibody, M720, is defined as specific for the IL-2 receptor because it blocks /sup 35/S-IL-2 binding to Con A blasts, reacts with lymphoblasts but not resting splenocytes, and inhibits IL-2 induced proliferation to mitogen, antigen, or allogeneic stimuli. Under appropriate culture conditions, the IL-2 receptor-specific antibody can act like IL-2 in that it will induce proliferation in T cells in the absence of additional antigen or mitogen. This agonistic effect is dependent on time, dose of antibody, and requires fetal calf serum (FCS) in the media. Because the FCS is not mitogenic by itself, the authors propose that the FCS components act as incomplete mitogen to induce appearance of IL-2 receptors but lack a factor which would push the majority of the cells into the S phase of the cell cycle. This factor is usually IL-2, but in the authors experiments, the IL-2 receptor-specific antibody can provide the same stimulus. These data indicate that factors like FCS can induce IL-2 receptors, but without additional IL-2 or receptor triggering, the cells will not proceed through the synthetic and proliferative phases of cell growth.

Eardley, D.D.; Makrides, V.



Diffusion of interleukin-2 from cells overlaid with cytocompatible enzyme-crosslinked gelatin hydrogels.  


In designing an implantable cell encapsulation construct to continuously deliver therapeutic proteins to a patient, it is critical that the biomaterial be compatible with the encapsulated cells, as well as conducive to the diffusion of desired molecules. As a continuation of our previous work, which demonstrated the cytocompatibility of gelatin hydrogels enzymatically crosslinked by microbial transglutaminase (mTG-gels), this work seeks to elucidate the diffusion properties that are needed for sustained release of therapeutic proteins produced by the engineered cells. HEK293 cells genetically engineered to secrete an anticancer drug, interleukin-2 (hIL2), through 4% mTG-gels used as a 1D diffusion model. Under steady-state conditions, cells secrete hIL2 at a therapeutic rate of 5.0-5.7 ng/cm(2)/h/10(6) cells. The diffusion coefficient of hIL2 through the hydrogels is D(m) = 4.0 x 10(-7) cm(2)/s. This value is comparable with similarly sized proteins through hydrogels and is further verified by modeling nonsteady-state diffusion through various thicknesses of the hydrogels, as well as by acellular diffusion chamber experiments. These findings demonstrate that the enzymatically crosslinked hydrogels are not only cytocompatible but also have suitable transport properties that will facilitate the design of sustained drug release devices. PMID:20740597

Yung, Chong Wing; Bentley, William E; Barbari, Timothy A



Effect of buparvaquone on the expression of interleukin 2 receptors in Theileria annulata-infected cells.  


Theileria annulata-infected cells were cultured in the presence or absence of human recombinant interleukin 2 (hrIL-2). This growth factor proved to be capable of enhancing the growth of the infected cells: its effect was marked, particularly when the cells were seeded at low densities, and it varied from cell line to cell line. The infected cells produced a factor that possessed the biological activities of IL-2, since their supernatants could enhance the proliferation of concanvalin A-stimulated (Con A) blasts. The reactivity of the parasitized cells to hrIL-2 was abolished following their treatment with the antitheilerial drug buparvaquone. In addition, the drug inhibited the binding of 125I-IL-2 to T. annulata-infected cells but failed to suppress its binding to Con A blasts. Northern blot analysis revealed that the drug had no effect on the expression of the alpha chain of the IL-2 receptor (IL-2R). Therefore, it is possible that buparvaquone interferes with the expression of the beta chain of the IL-2R. The role of IL-2 and the IL2R in the permanent proliferation of T. annulata-infected cells is discussed. PMID:1409527

Ahmed, J S; Rintelen, M; Schein, E; Williams, R O; Dobbelaere, D



Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide.  


The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment. PMID:11468348

Afonin, P V; Fokin, A V; Tsygannik, I N; Mikhailova, I Y; Onoprienko, L V; Mikhaleva, I I; Ivanov, V T; Mareeva, T Y; Nesmeyanov, V A; Li, N; Pangborn, W A; Duax, W L; Pletnev, V Z



Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide  

PubMed Central

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 ? resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen–antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly ?-helical conformation similar to that in the epitope fragment 64–72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody–antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the ?-helical character of the epitope fragment.

Afonin, Pavel V.; Fokin, Andrey V.; Tsygannik, Igor N.; Mikhailova, Irina YU.; Onoprienko, Lyudmila V.; Mikhaleva, Inna I.; Ivanov, Vadim T.; Mareeva, Tat'yana YU.; Nesmeyanov, Vladimir A.; Li, Naiyin; Pangborn, Walter A.; Duax3, William L.; Pletnev, Vladimir Z.



Employment of Oligodeoxynucleotide plus Interleukin-2 Improves Cytogenetic Analysis in Splenic Marginal Zone Lymphoma  

PubMed Central

To compare the efficiency of novel mitogenic agents and traditional mitosis inductors, 18 patients with splenic marginal zone lymphoma (SMZL) were studied. Three cultures using oligodeoxynucleotide (ODN) plus interleukin-2 (IL-2), or TPA, or LPS were setup in each patient. Seventeen/18 cases with ODN + IL2 had moderate/good proliferation (94, 4%) as compared with 10/18 cases with TPA and LPS (55%) (P = .015); 14/18 (77, 7%) cases with ODN + IL2 had sufficient good quality of banding as compared with 8/18 cases (44, 4%) with TPA and LPS. The karyotype could be defined from ODN + IL2-stimulated cultures in all 18 patients, 14 of whom (77, 7%) had a cytogenetic aberration, whereas clonal aberrations could be documented in 9 and in 3 cases by stimulation with LPS and TPA, respectively. Recurrent chromosome aberrations in our series were represented by aberrations of chromosome 14q in 5 patients, by trisomy 12 and 7q deletion in 4 cases each, and by abnormalities involving 11q and 13q in two cases each. These findings show that stimulation with ODN + IL2 offers more mitotic figures of better quality and results in an increased rate of clonal aberrations in SMZL, making this method ideal for prospective studies aiming at the definition of the prognostic impact of cytogenetic aberrations in this disorder.

Bardi, Antonella; Cavazzini, Francesco; Rigolin, Gian Matteo; Tammiso, Elisa; Volta, Eleonora; Pezzolo, Elisa; Formigaro, Luca; Sofritti, Olga; Daghia, Giulia; Ambrosio, Cristina; Rizzotto, Lara; Abass, Awad E.; D'Auria, Fiorella; Musto, Pellegrino; Cuneo, Antonio



Loss of Neuronal Phenotype and Neurodegeneration: Effects of T Lymphocytes and Brain Interleukin-2  

PubMed Central

Loss of neuronal phenotype and reversal of neuronal atrophy have been demonstrated in different models of central nervous system (CNS) injury. These processes may be generalizable to different types of brain neurons and circuitry. The idea that some injured neurons may lose their phenotype and/or atrophy with the potential to rejuvenate is a remarkable and potentially promising form of neuronal plasticity that is not well understood. In this paper, we present some of our laboratory’s basic neuroimmunology research showing that peripheral T cells entering the CNS, and brain-derived interleukin-2 (IL-2), play significant roles in these intriguing processes. Our findings suggest, for example, that T cell immunosenesence could be involved in related processes of brain aging and contribute to neurodegenerative disease. Neuroimmunological approaches may provide new insights into yet undiscovered factors and brain mechanisms that regulate changes in neuronal integrity associated with aging and disease. Such findings could have important implications for discovering more effective strategies for treating patients with neurotrauma and neurodegenerative diseases (e.g., Alzheimer’s disease).

Meola, Danielle; Huang, Zhi; Ha, Grace K; Petitto, John M



Systemic administration of interleukin-2 inhibits inflammatory neutrophil migration: role of nitric oxide  

PubMed Central

Interleukin-2 (IL-2) has proinflammatory properties that limit its therapeutic use. Its side effects are mainly explained by the induction of a vascular leakage syndrome. Cytokines, as TNF-? and IL-1?, and nitric oxide (NO) generated by IL-2-activated leukocytes play a role in this defect. As the systemic release of these mediators inhibits neutrophil migration to a specific inflammatory site, we investigated now whether IL-2 administrated systemically inhibits the neutrophil recruitment to the inflamed peritoneum. The involvement of NO in the process was also addressed. Using peritoneal neutrophils, we show that the intravenous treatment of the mice with IL-2 inhibits the neutrophil migration induced by carrageenin, LPS or fMLP. In confirmation, IL-2-treated mice showed a significant reduction in leukocyte rolling and adhesion in mesenteric microcirculation evaluated after carrageenin, LPS and fMLP injections. Aminoguanidine prevented the inhibitory effect of IL-2 on carrageenin-induced neutrophil migration, rolling and adhesion. In contrast, IL-2 failed to reduce the lung leukocyte infiltration induced by LPS. Therefore, IL-2 inhibition of neutrophil migration is organ specific. Our results indicate that IL-2 administered systemically inhibits neutrophil recruitment to some inflammatory sites through a mechanism dependent on NO. The results also reinforce the needs to determine the mechanism by which patients treated with IL-2 show increased risks of infection.

Moreno, Susana E; Alves-Filho, Jose C; Bertozi, Giuliana; Alfaya, Tais M; Theze, Jacques; Ferreira, Sergio H; Vargaftig, Bernardo Boris



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


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

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



CD4+CD25+ T Regulatory Cells, Immunotherapy of Cancer, and Interleukin-2  

PubMed Central

Summary: CD4+CD25+ T regulatory (Treg) cells control immunologic tolerance to self-antigens and play a role in suppressing antitumor immune responses, but the mechanism of suppression in vivo remains uncertain. Recently, signaling through the high-affinity interleukin-2 (IL-2) receptor has been shown to be critical for Treg cell differentiation and survival in vivo. Mice deficient in IL-2 or its receptor (CD25 or CD122) or deficient in downstream signaling molecules, including JAK-3 and STAT-5, do not develop a stable population of Treg cells and subsequently acquire lymphoproliferative disease and autoimmunity. in vitro, IL-2 is required to expand Treg cells and to induce their suppressive characteristics. Conversely, IL-2-based regimens can activate cellular antitumor immunity and are the mainstay of immunotherapies directed against melanoma and kidney cancers. Given the seemingly disparate effects of IL-2, the authors discuss the possibility that IL-2 may not be the optimal T-cell growth factor in vivo, but rather an inducer of self-tolerance. The authors propose that other ?c-signaling cytokines, including IL-15, may be alternative choices for the immunotherapy of cancer.

Antony, Paul Andrew; Restifo, Nicholas P



Role of interleukin-2 and herpes simplex virus 1 in central nervous system demyelination in mice.  


We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination. PMID:23986600

Mott, Kevin R; Zandian, Mandana; Allen, Sariah J; Ghiasi, Homayon



Direct effect of interleukin 2 on chronic lymphocytic leukaemia B cell functions and morphology.  

PubMed Central

The functional and morphological changes induced by recombinant interleukin 2 (IL-2) were studied in purified B cells from patients with untreated B chronic lymphocytic leukaemia (B-CLL). In eight of nine patients, purified B-CLL cells increased their DNA synthesis in response to IL-2 without preactivation in vitro. This response, studied in detail in three patients, was dose dependent and reached a maximum on day 5 or 6. IL-2 induced or increased IgM secretion in cultures from five of the nine patients studied. Two of this responsive group were particularly interesting as IL-2 not only stimulated IgM secretion but also induced the secretion of IgG. Immunoglobulin production was invariably monoclonal. B CLL cells incubated with IL-2 showed distinct morphological changes including an increase in the size of cytoplasm and enlargement of nuclei together with the appearance of nucleoli. These changes were present in all IL-2 treated cultures but were more pronounced in those containing immunoglobulin secreting cells. None of the IL-2 induced changes appeared to correlate with the clinical stage of the disease or the level of Tac antigen expression on the freshly isolated CLL B cells. Images Fig. 2

Malkovska, V; Murphy, J; Hudson, L; Bevan, D



Inhibition of interleukin-2 production by adherent cell factors from lepromatous leprosy patients.  

PubMed Central

Twenty-four hour supernatants (MoF) were obtained from monocyte rich 2 h adherent cells of 19 leprosy patients and four healthy contacts. MoF from borderline and lepromatous patients produced 52-61% inhibition of human interleukin-2 (IL-2) production by a PHA conditioned T cell line (Jurkat). Non-adherent cell supernatants and MoF from tuberculoid and healthy individuals had little effect on IL-2 production. The suppression effected by MoF was in the first 12 h of initiation of PHA stimulated Jurkat cell cultures. Suppressive MoF did not interfere with (1) IL-2 release, (2) IL-2 utilization by Con A-induced T cell blasts or (3) constitutive proliferation of Jurkat cells. Such MoF were released spontaneously from adherent cells of bacilliferous leprosy patients but required in vitro antigen triggering in long term treated lepromatous patients. It is possible that the unresponsiveness associated with lepromatous leprosy is related to the inhibition of IL-2 production by suppressive factors, thereby, preventing the further expansion of antigen reactive T cells.

Nath, I; Jayaraman, J; Sathish, M; Bhutani, L K; Sharma, A K



Evaluation of the cardiovascular toxic effect of recombinant interleukin-2 in rats.  


Cardiac and vascular toxicity of recombinant interleukin-2 (rIL-2) was evaluated by in vitro and in vivo experiments in rats. Using isolated spontaneously beating atria, no changes were detected in heart rate or myocardial contractility in response to rIL-2 treatment at concentrations ranging from 0.1-100 U/ml. Daily sc administration of rIL-2 for 7 consecutive days (at doses of 2.3 X 10(4) to 1.15 X 10(6) U/ml) produced a prolongation of the QaT interval and significant modifications in serum electrolyte concentrations. Ex vivo contractility of atria excised at the end of rIL-2 treatment showed no deterioration in myocardial contractility with increasing dosage. rIL-2, at concentrations of 0.1-1000 U/ml, did not induce any modification of perfusion pressure in isolated rat tail artery but produced a significant displacement to the left of the dose-response curves for norepinephrine compared to basal conditions. The relative potencies were not dose-dependent. Our results indicate that rIL-2 does not exert a direct cardiotoxic effect, and suggest an indirect action on vascular smooth muscle, i.e., an aspecific modulation of vascular response to mediators. PMID:2285243

Favalli, L; Lanza, E; Rozza, A; Galimberti, M; Villani, F


Low and high affinity cellular receptors for interleukin 2. Implications for the level of Tac antigen  

PubMed Central

Interleukin 2 promotes proliferation of T cells by virtue of its interaction with a high-affinity cell surface receptor. This receptor is a 55,000 mol wt glycoprotein that is also recognized by the murine monoclonal antibody, anti-Tac. Quantitative binding studies with radiolabeled IL-2 and anti-Tac, however, initially indicated far more antibody binding sites per cell than IL-2 binding sites. Extension of the IL-2 binding analysis to concentrations several thousand-fold higher than that necessary for the T cell proliferative response demonstrated the existence of a class (or classes) of low-affinity IL-2 binding sites. Inclusion of the low-affinity IL-2 binding greatly reduced the quantitative discrepancy in the ligand binding assays. That the low-affinity binding, as well as the high-affinity interaction, was associated with the Tac molecule was indicated by the finding that the antibody could substantially or totally block the entire spectrum of IL- 2 binding and by the finding that IL-2 could in turn block all radiolabeled anti-Tac binding. The low-affinity sites were found on activated T cells, several human and murine T cell lines and two examples of Tac-positive B cells. The physiological role of the low- affinity IL-2 binding sites and the molecular changes in the Tac protein that give rise to the affinity differences remain open to investigation.



Early precursor thymocytes can produce interleukin 2 upon stimulation with calcium ionophore and phorbol ester  

SciTech Connect

T-cell precursors were stimulated with a conventional T-cell mitogen or with the calcium ionophore A23187 in order to determine whether pre-T cells acquire the ability to produce interleukin 2 (IL-2) before they acquire the ability to respond to antigen or mitogenic lectins. Immature T cells were obtained by eliminating mouse thymocytes that expressed the Lyt2 and L3T4 cell surface proteins. The remaining Lyt2/sup -/, L3T4/sup -/ cells were stimulated for IL-2 production by using concanavalin A (Con A) or A23187, together with phorbol 12-myristate 13-acetate (PMA). The authors found that these double-negative thymocytes were unresponsive to Con A plus PMA but produced substantial amounts of IL-2 when stimulated with A23187 plus PMA. In contrast, both stimulation regimens induced more mature T-lymphocyte populations to produce IL-2. This implies that developing T cells acquire the ability to make IL-2 upon induction before they acquire the ability to be triggered by Con A. Day-15 fetal and cortical thymocytes were also tested for their ability to make IL-2. Both populations failed to synthesize this growth factor, even when stimulated with A23187 and PMA. For cortical thymocytes, this result, together with the finding that A23187 plus PMA fails to activate these cells, suggests that this population is immunologically inert rather than immature.

Lugo, J.P.; Krishnan, S.N.; Sailor, R.D.; Rothenberg, E.V.



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

SciTech Connect

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.

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



Human interleukin 2 receptor. beta. -chain gene: Chromosomal localization and identification of 5 prime regulatory sequences  

SciTech Connect

Interleukin 2 (IL-2) binds to and stimulates activated T cells through high-affinity IL-2 receptors (IL-2Rs). Such receptors represent a complex consisting of at least two proteins, the 55-kDa IL-2R{alpha} chain and the 70-kDa IL-2R{beta} chain. The low-affinity, IL-2R{alpha} chain cannot by itself transduce a mitogenic signal, whereas IL-2 stimulates resting lymphocytes through the intermediate-affinity, IL-2R{beta} receptor. The authors report here identification of the genomic locus for IL-2R{beta}. The exons are contained on four EcoRI fragments of 1.1, 9.2, 7.2, and 13.7 kilobases. The 1.1-kilobase EcoRI fragment lies at the 5{prime}-most end of the genomic locus and contains promoter sequences. The promoter contains no TATA box-like elements but does contain the d(GT){sub n} class of middle repetitive elements, which may play an interesting regulatory role. The IL-2R{beta} gene is localized to chromosome 22q11.2-q12, a region that is the locus for several lymphoid neoplasias.

Gnarra, J.R.; Otani, Hiroki; Wang, M.G.; McBride, O.W.; Sharon, M.; Leonard, W.J. (National Institutes of Health, Bethesda, MD (USA))



Enhanced cytotoxic response of natural killer cells to interleukin-2 in Alzheimer's disease.  


Experimental data suggest an involvement of immune cellular components in the development of Alzheimer's disease (AD). Against this background, the spontaneous natural killer (NK) cell activity and the NK-induced cytotoxicity after interleukin-2 (IL-2) were studied in healthy elderly subjects and in patients with dementia of Alzheimer type (SDAT) and multi-infarct type (MID). Higher NK cytotoxicity (expressed as total lysis and percent increase) at different IL-2 concentrations (50 and 100 IU/ml/cells) was demonstrated in patients with SDAT than in healthy elderly subjects (p < 0.001) and MID patients (p < 0.001). NK cell activity of MID patients was similar to that of healthy elderly and healthy young subjects. A negative correlation between the percent increase in NK cytotoxicity after IL-2 and the Mini Mental State Examination Score was also found in SDAT patients (p < 0.01). Alterations of IL-2-mediated NK cytotoxicity may therefore support the neuroimmune hypothesis of AD. PMID:8915041

Solerte, S B; Fioravanti, M; Severgnini, S; Locatelli, M; Renzullo, M; Pezza, N; Cerutti, N; Ferrari, E


A novel approach to purging of leukemia by activation of bone marrow with interleukin 2.  


The cytotoxic potential of interleukin 2 (IL-2) activated bone marrow (ABM) was compared with that of IL-2 activated peripheral blood lymphocytes (LAK cells) against three hematologic tumor cell lines (K-562, CEM, Daudi) and fresh lymphoid blasts in short-term chromium release assays. ABM was found to be superior to LAK cells against all tumor cells tested. The recovery of bone marrow (BM) cells dropped with passage of time in culture but their clonogenic potential was not impaired (with or without IL-2). BM contaminated with CEM cells and treated with IL-2 showed significant ability to purge itself of the leukemic cells in semisolid agar culture; the purging ability of 3- and 1-day ABM was comparable. IL-2 alone or BM alone had no influence on the growth of CEM cells. This study suggests that BM can be activated with IL-2 in vitro to generate the ability to eliminate contaminating leukemic cells without affecting its progenitor cell function in vitro. PMID:2252959

Charak, B S; Malloy, B; Agah, R; Mazumder, A



Effects of sleep and sleep deprivation on catecholamine and interleukin-2 levels in humans: clinical implications.  


The objective of this study was to evaluate the effects of nocturnal sleep, partial night sleep deprivation, and sleep stages on catecholamine and interleukin-2 (IL-2) levels in humans. Circulating levels of catecholamines and IL-2 were sampled every 30 min during 2 nights: undisturbed, baseline sleep and partial sleep deprivation-late night (PSD-L; awake from 0300-0600 h) in 17 healthy male volunteers. Sleep was monitored somnopolygraphically. Sleep onset was associated with a significant (P < 0.05) decline of circulating concentrations of norepinephrine and epinephrine, with a nocturnal nadir that occurred 1 h after nocturnal sleep. On the PSD-L night, levels of norepinephrine and epinephrine significantly (P < 0.05) increased in association with nocturnal awakening. During stage 3-4 sleep, levels of norepinephrine, but not epinephrine, were significantly lower (P < 0.05) compared to average levels during the awake period, stages 1-2 sleep, and rapid eye movement sleep. Nocturnal levels of circulating IL-2 did not change with sleep onset or in relation to PSD-L or the various sleep stages. We conclude that sleep onset is associated with changes in levels of circulating catecholamines. Loss of sleep and disordered sleep with decreases in slow wave sleep may serve to elevate nocturnal catecholamine levels and contribute to cardiovascular disease. PMID:10372697

Irwin, M; Thompson, J; Miller, C; Gillin, J C; Ziegler, M



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

PubMed Central

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

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



Effect of 1,1-dimethylhydrazine on lymphoproliferation and interleukin 2 immunoregulatory function.  


The studies reported here suggest that the immunomodulatory effects of 1,1-dimethylhydrazine (UDMH) are associated, in part, with interference with interleukin 2 (IL-2) regulatory action. Concanavalin A (Con A)-stimulated DNA synthesis in murine splenocytes was inhibited from 18.6 to 44.1% at sub-toxic concentrations of UDMH (10 to 50 micrograms/ml) and IL-2-dependent DNA synthesis in CTLL-20 cells was inhibited from 11.3 to 41.58% at sub-toxic concentrations of UDMH (10 to 50 micrograms/ml). In addition, UDMH suppressed phorbol myristic acetate (PMA)-stimulated IL-2 production in EL-4 cells by up to 30% and slightly suppressed IL-2 production by Con A-stimulated murine splenocytes. In all cases, inhibition was evident at sub-toxic UDMH concentrations and was demonstrated to be independent of inactivation of IL-2 or interference with IL-2 absorption. It is suggested that UDMH has the potential to modify immune function through interference with IL-2 production and especially the lymphoproliferative response to IL-2. PMID:2331149

Bauer, R M; Tarr, M J; Olsen, R G


Inhibition of nociceptive withdrawal reflex by microinjection of interleukin 2 into rat locus coeruleus.  


This study was to examine the effects of microinjection of human recombinant interleukin 2 (IL-2) into locus coeruleus (LC) on spinal nociception. Following application of IL-2 (0.1 microl, 10 pM) into LC, the percentage of inhibition of nociceptive C responses of reflex at 3, 9, 15, 21 and 27 min after injection were 88.2 +/-9.4%, 84.0 +/- 11.8%, 89.7 +/- 10.5%, 57.1 +/- 8.7% and 26.3 +/- 12.2%, respectively. Also, the expression of Fos protein in superficial dorsal horn was reduced by 73.01 +/- 13.58% of control (P<0.0001). Naloxone (10 microg, i.p.) completely blocked the IL-2-induced inhibition of C responses. The results clearly show that IL-2 receptors present in LC mediate descending inhibition of the spinal nociception, which may couple with the activation of opioid receptors on LC neurons. PMID:11021983

Guo, H; Zhao, Z Q


Enhancement of a delayed hypersensitivity reaction to a contact allergen, by the systemic administration of interleukin-2.  

PubMed Central

The immunopharmacological effects of interleukin-2 (IL-2) on the sensitization and effector phases of the delayed-type hypersensitivity (DTH) reaction were studied using contact sensitivity to the haptenizing agent dinitrochlorobenzene (DNCB). When administered at the time of priming to DNCB, IL-2 had no effect on the subsequent magnitude of the response. Interleukin-2 was, however, able to increase the magnitude of the response when given at the time of secondary challenge; the degree of change was directly related to the dose of IL-2. The proportions of T cells in the draining lymph node and spleen of IL-2-treated animals decreased by approximately one-third, but there was no alteration to the balance between CD4+ and CD8+ T cells. The results suggest that the increase in DTH observed was due to a pharmacological effect rather than to an increase in T-cell number.

Zaloom, Y; Walsh, L P; McCulloch, P; Gallagher, G



Characterization of the interleukin 2 receptor beta chain using three distinct monoclonal antibodies.  

PubMed Central

The human high-affinity receptor for interleukin 2 (IL-2) has been proposed as being a membrane complex composed of at least two distinct polypeptide chains: p55 (alpha chain), recognized by the anti-Tac monoclonal antibody (mAb), and p75 (beta chain), both of which are capable of binding IL-2. Whereas the alpha chain itself has been shown to be nonfunctional, the beta chain appears to be pivotal in the IL-2 signal transduction, although the beta chain is otherwise poorly characterized. Three beta chain-specific mAbs, designated Mik-beta 1, -beta 2, and -beta 3, were developed. Mik-beta 1 and -beta 2 completely inhibited the IL-2 binding to the beta chain, whereas Mik-beta 3 immunoprecipitated the beta chain crosslinked with 125I-labeled IL-2. The beta chain immunoprecipitated by these mAbs was revealed to have a Mr of 68,000-72,000. High-affinity IL-2 binding was completely abolished by Mik-beta 1. Although IL-2-dependent T-cell growth at high IL-2 concentrations was not inhibited by the anti-Tac, it was almost completely inhibited by Mik-beta 1 in the presence of the anti-Tac. These results clearly indicate that the beta chain is an indispensable component to the high-affinity IL-2 receptor and is responsible for the IL-2 signal transduction. The beta chain was found to be constitutively expressed without the alpha chain on the surface of peripheral blood Leu-19+ natural killer cells. Images

Tsudo, M; Kitamura, F; Miyasaka, M



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

NASA Astrophysics Data System (ADS)

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

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



Suppression of multiple bioactivities of interleukin-1 and interleukin-2 production by U937 conditioned medium  

SciTech Connect

The human macrophage-like cell line U937 spontaneously produces a factor which blocks interleukin-1 (IL-1) activity for mouse thymocytes but not mitosis of T-lymphoblastoid cells. The authors examined the effects of U937 conditioned medium (CM) on other IL-1 activities and on interleukin-2 (IL-2) production. U937 was cultured at 5 x 10/sup 6//ml in RPMI-1640 at 37/sup 0/C for 5 days. The resulting CM inhibited the mitogenic response of C3H/HeJ mouse thymocytes to an IL-1 standard, with an inhibitory of activity of 6.64 U/ml (1 U = reciprocal dilution producing 50% inhibition of maximal response). Similarly, CM inhibited (10.12 U/ml) the fibroblast stimulation promoter activity of IL-1. The effect of CM on IL-2 production was assessed in a direct assay in which IL-2 production by ..gamma..-irradiated (12,000 rads) MLA-144 lymphosarcoma cells was assayed as /sup 3/H-thymidine incorporation in CTLL-20 cells. The suppressive activity of CM was 4.95 U/ml; CM did not interfere with the response of CTLL-20 to IL-2. These studies establish that U937 produces factors with multiple, related biological activities; U937 CM blocks IL-2 dependent (thymocyte mitogenesis) and IL-2 independent (fibroblast proliferation) IL-1 activities and interferes with production of, but not response to, IL-2. U937 is an excellent model to study growth inhibitory properties of mononuclear phagocytes.

Wiblin, R.T.; Edmonds, K.; Ellner, J.J.



The interleukin-2 receptor in lesions and serum of bullous pemphigoid.  


The interleukin-2 receptor (IL-2R) is mainly expressed on activated T cells. Depending on its rate of synthesis, a portion is released from the cell surface as soluble IL-2R (sIL-2R). Since the role of mononuclear cells in the pathology of bullous pemphigoid (BP) is not well understood, we determined the sIL-2R in both blister fluid and serum of 15 BP patients with generalized disease before initiating systemic treatment. In addition, we obtained both lesional and perilesional skin biopsies and examined the mononuclear infiltrate with a panel of monoclonal antibodies. In BP blisters, sIL-2R levels were significantly increased (2070 +/- 350 U/ml), (+/- SEM) compared with serum samples taken at the time of blister puncture (1340 +/- 290 U/ml). In six patients with blisters due to second-degree burns or friction and in five suction blister volunteers, sIL-2R levels were normal in both blisters and serum. In BP, elevated serum levels decreased to normal during therapy, correlating with disease activity. The immunohistology showed that 30% of mononuclear cells in the dermal infiltrate of lesional skin expressed the IL-2R, whereas only 15% were positive in perilesional skin. IL-2R-positive cells are the most likely source of the shed receptor in BP blisters. Our results indicate the presence of activated T cells in lesions and peripheral blood of BP and thus underline the importance of cell-mediated immune mechanisms in the pathology of this disease. PMID:1503497

Zillikens, D; Ambach, A; Schuessler, M; Dummer, R; Hartmann, A A; Burg, G



A novel approach to immunomodulation of frozen human bone marrow with interleukin-2 for clinical application.  


Interleukin-2 (IL-2) activation of fresh or frozen bone marrow (BM) in vitro generates killer cells with potent anti-tumor effect both in vitro and in vivo. The IL-2-activated BM (ABM) retains the capacity to reconstitute the hematopoietic system in an autologous bone marrow transplantation (ABMT) setting. The killer cells lose their cytotoxicity if the ABM undergoes the procedures of freezing and thawing. Therefore, for clinical application, the ABM has to be generated after thawing a frozen stock of BM before ABMT. The thawed BM cells are fragile and may undergo lysis, resulting in clump formation and cell loss. The frozen autograft also contains components of cryoprotectant mixture whose effects on the generation of ABM have not been defined. The present studies have been carried out to optimize a technique of handling the frozen BM for immunomodulation with IL-2 for 24 h at 37 degrees C prior to ABMT, with minimal loss of cells. IL-2-activation of BM was carried out in bags containing serum free medium which were designed to permit gaseous exchange. Addition of deoxyribonuclease (DNAse) (100 micrograms/ml of BM concentrate) immediately after thawing and the presence of heparin (20 units/ml) in the medium completely abrogated immediate or delayed clumping of cells. The presence of DNAse and/or heparin during in vitro culture did not affect the cell viability, cytotoxicity against tumor cells or the progenitor cell activity of the ABM; all these functions were well maintained even when BM was placed in culture immediately after thawing (without washing). There was no microbial contamination.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8435664

Charak, B S; Areman, E M; Dickerson, S A; Choudhary, G D; Sacher, R; Kotula, P L; Brown, E G; Mazumdar, A



Fibrinolysis, thrombocytopenia, and coagulation abnormalities complicating high-dose interleukin-2 immunotherapy.  


High-dose interleukin-2 (IL-2) immunotherapy can cause hypotension, respiratory distress, interstitial edema, and thrombocytopenia, similar to endotoxic shock. We have observed that IL-2 has no direct effect on coagulation factors in vitro, but it has been observed to alter the coagulant properties of vascular endothelium. Accordingly, we investigated the possibility that IL-2 infusions initiate plasma fibrinolysis and disseminated intravascular coagulation (DIC). We studied the clinical course, platelet count, and coagulation profile in response to IL-2 infusion in seven patients, two with metastatic melanoma and five with metastatic renal cell carcinoma. Every patient experienced hemodynamic instability and thrombocytopenia, and one patient suffered an unusual complication, mesenteric thrombosis. No patient had appreciable changes in the prothrombin time or the partial thromboplastin time, nor did factors V or VIII decline in the two patients observed. In four patients examined, we found decreased titers of Hageman factor (factor XII), high molecular weight kininogen, prekallikrein, and plasma thromboplastin antecedent, as if these had been consumed by reactions of the intrinsic pathway of thrombin formation. Circulating D-dimer fragments were found in the plasma of every patient at some point during each infusion cycle, and we observed decreased titers of plasminogen in the four patients just mentioned, suggesting that IL-2 infusions initiated fibrinolysis. Taken together, the clotting factor derangements and related toxicity phenomena cannot be ascribed firmly to DIC. Activation of the intrinsic (contact) system of coagulation, however, may provide one link between the vascular endothelial surface alterations caused by IL-2 infusions and the development of the systemic toxicity that resembles septic shock. PMID:1987312

Fleischmann, J D; Shingleton, W B; Gallagher, C; Ratnoff, O D; Chahine, A



Increased incidence of hypersensitivity to iodine-containing radiographic contrast media after interleukin-2 administration.  


Eight of 28 (28%) cancer patients with liver metastases treated by either splenic (four) or hepatic (four) arterial infusion of recombinant interleukin-2 (rIL-2) developed hypersensitivity reactions to iodine-containing radiographic contrast media. These reactions consisted of fever, chills, malaise, nausea and vomiting, skin rash, diarrhea, and occasionally, hypotension. Reactions usually occurred 1 month after the initial arteriographic procedure and rIL-2 infusion, with 1-hour to 4-hour intervals between procedure and reexposure of the patient to the iodine-containing contrast medium (used in conjunction with computerized tomography or repeated arteriography for subsequent courses of rIL-2 infusions) and the onset of symptoms. Prompt administration of corticosteroids during the reaction and premedication of patients who were known to have had a reaction in the past were very effective in stopping reactions or preventing them from reoccurring. The high incidence (28%) of hypersensitivity reactions, the temporal relationship (4 hours) between the arteriographic procedure (utilizing iodine-containing contrast medium) and the initial infusion of rIL-2 (while some of the contrast medium was still present), and the absence of such hypersensitivity reactions among patients receiving systemic (intravenous) rIL-2 (not requiring the use of concomitant iodine-containing contrast medium) provide additional evidence that in the presence of a potentially immunogenic moiety, rIL-2, a potent stimulant of the human immune system, can produce an initial sensitization followed by subsequent anamnestic reaction upon reexposure of the patient to the immunogen (even without the additional rIL-2). PMID:2311064

Zukiwski, A A; David, C L; Coan, J; Wallace, S; Gutterman, J U; Mavligit, G M



High-dose Interleukin-2 for the Treatment of Metastatic Renal Cell Carcinoma  

PubMed Central

BACKGROUND The treatment of metastatic renal cell carcinoma (RCC) with high-dose interleukin-2 (HD IL-2) has resulted in durable tumor regression in a minority of patients. The current study presents the authors’ 20-year experience administering this immunotherapeutic agent. METHODS Patients with metastatic RCC (n = 259) were treated with HD IL-2 alone from January 13, 1986 through December 31, 2006 at the Surgery Branch of the National Cancer Institute. Potential predictive factors for response and survival, both pretreatment and treatment-related, were first subjected to univariate analysis and then to multivariate logistic regression or a Cox proportional hazards model. Finally, the authors investigated Memorial Sloan-Kettering Cancer Center (MSKCC) prognostic factors for survival to assess their predictive value in the patient population in the current study. RESULTS A total of 23 patients experienced a complete response and 30 patients achieved a partial response, for an overall objective response rate of 20%. All partial responders had developed disease recurrence at the time of last follow-up, but only 4 complete responders had experienced disease recurrence by that time. Despite toxicities, only 2 patients developed treatment-related mortalities over this same time period. A higher baseline weight (P =.05) and MSKCC prognostic factors (P = .02) were found to be the variables most associated with response. For survival >4 years and overall survival, several pretreatment and treatment-related factors maintained significance, but none more so than response (P <.0001). CONCLUSIONS HD IL-2 can induce complete tumor regression in a small number of patients, and many patients have experienced extended disease-free intervals. Given its relative safety, HD IL-2 should still be considered a first-line therapy in patients with metastatic RCC who have an overall good performance status.

Klapper, Jacob A.; Downey, Stephanie G.; Smith, Franz O.; Yang, James C.; Hughes, Marybeth S.; Kammula, Udai S.; Sherry, Richard M.; Royal, Richard E.; Steinberg, Seth M.; Rosenberg, Steven



Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells.  

PubMed Central

p56lck, a member of the src family of cytoplasmic tyrosine kinases, is expressed predominantly in T cells where it associates with the T-cell surface molecules CD4 and CD8. Mutants of CD4 and CD8 that have lost the ability to associate with p56lck no longer enhance antigen-induced T-cell activation. This suggests that p56lck plays an important role during T-cell activation. In an effort to understand the function of p56lck in T cells, a constitutively activated lck gene (F505lck) was introduced into T-helper hybridoma cell lines by retroviral infection. In four T-cell lines we examined, the activated lck protein stimulated interleukin-2 (IL-2) production, a hallmark of T-cell activation, in the absence of antigenic stimulation. In addition, a marked increase in antigen-independent IL-2 production was apparent when T cells infected with a temperature-sensitive F505lck were shifted to the permissive temperature. Only one cell line expressing F505lck exhibited increased sensitivity to antigenic stimulation. The SH3 domain of p56lck was dispensable for the induction of antigen-independent IL-2 production. In contrast, deletion of the majority of the SH2 domain of p56F505lck reduced its ability to induce spontaneous IL-2 production markedly. Activated p60c-src also induced antigen-independent IL-2 production, whereas two other tyrosine kinases, v-abl and the platelet-derived growth factor receptor, did not. Tyrosine phosphorylation of a 70-kDa cellular protein was observed after cross-linking of CD4 in T cells expressing F505lck but not in cells expressing F527src. Images

Luo, K; Sefton, B M



Local versus systemic interleukin-2: Tumor formation by wild-type and B7-1-positive murine melanoma cells  

Microsoft Academic Search

Modification of murine K1735 melanoma cells to express the immune costimulator B7-1 had no effect on tumor formation in syngeneic mice. In contrast, <40% of mice inoculated with K1735 cells modified to secrete murine interleukin-2 (IL-2) formed tumors, and no tumors formed when the K1735 cells coexpressed both murine IL-2 and B7-1. However, administration of systemic recombinant human IL-2 had

Amanda L Barnard; Farzin Farzaneh; Joop Gäken; David Darling



Recovery from autoimmunity of MRL\\/lpr mice after infection with an interleukin-2\\/vaccinia recombinant virus  

Microsoft Academic Search

INTERLEUKIN-2 (IL-2) is a T-cell derived molecule implicated in the clonal expansion of antigen-activated T cells1 and in T-cell development2. IL-2 is also implicated in autoimmune disease3, although its role is still controversial4,5. Murine systemic lupus erythematosus (SLE) is a good model for human SLE as most of the immunological abnormalities in the human disease also seem to be operative

Jose C. Gutierrez-Ramos; Jose L. Andreu; Yolanda Revilla; Eladio Viñuela; Carlos Martinez-A



Immunochemotherapy with interleukin-2, interferon- ? and 5-fluorouracil for progressive metastatic renal cell carcinoma: a multicenter phase II study  

Microsoft Academic Search

In patients with metastatic renal cell carcinoma response rates of 7–26% have been achieved with immunotherapy. A high response rate of 48% in 35 patients has been reported for treatment with the combination of interferon-? (IFN-?), interleukin-2 (IL-2) and 5-fluorouracil (5-FU) (Atzpodien et al (1993 a) Eur J Cancer 29A: S6–8). We conducted a multicentre phase II study to confirm

C M L van Herpen; R L H Jansen; W H J Kruit; K Hoekman; G Groenewegen; S Osanto; P H M De Mulder



Patients with condyloma acuminatum exhibit decreased interleukin-2 and interferon gamma production and depressed natural killer activity  

Microsoft Academic Search

Peripheral blood mononuclear cells were obtained from 20 untreated condyloma acuminatum patients and from an equal number of sex- and age-matched controls and assayed for cell surface antigen expression, natural killer activity, and lymphokine production. Patient peripheral blood mononuclear cells had significantly lower helper-to-suppressor T-cell ratios (Leu3\\/Leu2) (pPPPin vitro production of interleukin-2 and interferon gamma and the percentage of Leu

Roberto Cauda; Stephen K. Tyring; Carlo E. Grossi; Arabella B. Tilden; Kenneth D. Hatch; W. Mitchell Sams; Samuel Baron; Richard J. Whitley



Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2  

Microsoft Academic Search

Incubation of resting lymphoid cells with recombinant interleukin 2 (IL-2) in vitro leads to the generation of lymphokine activated killer (LAK) cells capable of lysing fresh tumor cell suspensions in short-term chromium-release assays. Previous studies have demonstrated that the injection of LAK cells plus low doses of recombinant IL-2 were capable of inhibiting the growth of pulmonary metastases. The authors

S. A. Rosenberg; J. J. Mule; P. J. Spiess; C. M. Reichert; S. L. Schwarz



Treatment of pulmonary metastases from kidney cell carcinoma with inhalational interleukin-2.10-year experience Hamburger Unicenter  

Microsoft Academic Search

Summary  \\u000a Systemic immunotherapy, notably with interleukin-2 (IL-2) and interferon-? (IFN?), has yielded a response rate of 10 % to\\u000a 30 % in metastic renal cell carcinoma. However, systemic immunotherapy is limited by severe side effects, and long-lasting\\u000a response is rare. Tumor palliation and quality-of-life are important end points for evaluating the clinical benefits of immunotherapy.\\u000a Experimental and clinical treatment models

H. Heinzer; E. Huland; M. Aalamian; H. Huland



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

Microsoft Academic Search

Summary Incubation of human lymphocytes with recombinant interleukin-2 (rIL-2) results in the generation of lymphokine-activated killer (LAK) cells capable of lysing a wide variety of tumor cells. The present study was undertaken to examine the effect of recombinant ? interferon (rIFN-?) on LAK cell cytotoxicity generated from different peripheral blood mononuclear cell (PBMC) subpopulations. When unseparated PBMC were stimulated by

Michel Toledano; Claire Mathiot; Jean Michon; Georges Andreu; Danielle Lando; Maud Brandely; Wolf H. Fridman



Effects of Interleukin2 on the Mechanical Restitution and Post-rest Contraction in Rat Ventricular Papillary Muscle  

Microsoft Academic Search

To determine whether application of interleukin-2 (IL-2) alters function of sarcoplasmic reticulum (SR), we measured mechanical restitution and post-rest potentiation (PRP) in isolated rat papillary muscles. Mechanical restitution curves were constructed by interpolating extrasystoles at different test intervals following a train of steady state beats. In control group, the maximal PRP was reached after 60-120s of rest and the maximal

G.-H. Lin; H.-L. Ru; P.-L. Wu; C.-M. Cao; Q. Xia



Improvement of Hepatitis B Virus DNA Vaccines by Plasmids Coexpressing Hepatitis B Surface Antigen and Interleukin2  

Microsoft Academic Search

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus




Soluble interleukin-2 receptor and soluble CD8 antigen levels in serum from patients with non-resectable lung cancer  

Microsoft Academic Search

Summary In a preliminary longitudinal study two women with histologically verified adenocarcinoma of the lung, without simultaneous infectious or inflammatory conditions, were seen every 2 weeks until death. In one of the patients serum soluble interleukin-2 receptor (sIL-2R) levels rose progressively while the levels for the other patient increased during the second half of the observation period. Serum soluble CD8

Jette Vibe-Petersen; Niels Tvede; Marcus Diamant; Anne Arnt Kjerulff; Hans Rahbek Sørensen; Vagn Andersen



Leukocyte orchestration in blood and tumour tissue following interleukin-2 based immunotherapy in metastatic renal cell carcinoma  

Microsoft Academic Search

With the objective of evaluating leukocyte orchestration in situ, serial blood samples and tumour tissue core needle biopsies were obtained at baseline and repeated after 1 month of therapy, among 49 consecutive single-institution patients with metastatic renal cell carcinoma (mRCC). Patients were treated with outpatient low-dose subcutaneous interleukin 2 (IL-2) and interferon a (IFN-a) alone ( n=23) or in combination with

Frede Donskov; Karen Marie Bennedsgaard; Marianne Hokland; Niels Marcussen; Rune Fisker; Hans Henrik Torp Madsen; Kirsten Fode; Hans von der Maase



SOCS-3 Is Tyrosine Phosphorylated in Response to Interleukin2 and Suppresses STAT5 Phosphorylation and Lymphocyte Proliferation  

Microsoft Academic Search

Members of the recently discovered SOCS\\/CIS\\/SSI family have been proposed as regulators of cytokine signaling, and while targets and mechanisms have been suggested for some family members, the precise role of these proteins remains to be defined. To date no SOCS proteins have been specifically implicated in interleukin-2 (IL-2) signaling in T cells. Here we report SOCS-3 expression in response




Combination Nonviral Interleukin 2 Gene Therapy and External-Beam Radiation Therapy for Head and Neck Cancer  

Microsoft Academic Search

Objectives: To demonstrate that the combination of nonviral murine interleukin 2 (mIL-2) gene therapy and external-beam radiation therapy (XRT) have an en- hanced therapeutic effect for the treatment of head and neck squamous cell carcinoma (HNSCC) in an ortho- topic murine model and to elucidate the mechanism of action. Methods: Randomized, controlled studies in the mu- rine orthotopic model of

David Bray; Shu-Zhen Yu; Hilary Koprowski; Juong Rhee; Sanjeev Kumar; Federica Pericle; Mohan Suntharalingam; David A. Van Echo; Daqing Li; Bert W. O'Malley



Effects of two basidiomycete species on interleukin 1 and interleukin 2 production by macrophage and T cell lines  

Microsoft Academic Search

Two basidiomycete species, Lentinus edodes mycelia (LEM) and Cordyceps sinensis (CS) were examined for induction of cytokines in murine macrophage cell line R309 (R309) and T cell line LBRM-33 1A5 (1A5). When lipopolysaccharide (LPS)-activated R309 were exposed to the extracts of basidiomycetes, R309 induced significant levels of interleukin 1 (IL-1). Interleukin 2 (IL-2) induction was recognized in 1A5 cultures in

Takashi Kawanishi; Yurika Ikeda-Dantsuji; Ariaki Nagayama



Serum soluble interleukin-2 receptor levels in patients with chronic hepatitis B virus infection and its relation with anti- HBc  

Microsoft Academic Search

AIM: To investigate the relationship between serum soluble interleukin-2 receptor (sIL-2R) level and anti-HBc in patients with chronic hepatitis B virus (HBV) infection. METHODS: Sera from 100 patients with chronic HBV infection and 30 healthy controls were included in this study. The patients were divided into group A (HBsAg (+), HBeAg (+) and anti-HBc (+), n = 50) and group

Ping Xiao; Qing-Feng Chen; Yan-Ling Yang; Zhen-Hua Guo; Hong Chen Ping Xiao


[Immunomodulating activity of natural killers in patients with breast tumors using vasopressin and interleukin 2 in vitro].  


The study included 10 female donors, 12 patients with benign and 59 with malignant tumors of the breast at various stages before and after treatment. The immunomodulating effect of vasopressin and interleukin-2 on blood-natural killer functional activity was studied in vitro. Vasopressin dose of 4 x 10(-1) IU/5 x 10(5) cells exerted an immunosuppressive effect while 4 x 10(-5) IU/5 x 10(5) cells stimulated immunity. The stimulating effect of optimal interleukin-2 dosage (20-40 U/5 x 10(5) cells) on natural killer functional activity appeared 1.5-2-times higher than that optimal vasopressin dose (4 x 10(-5)/5 x 10(5) cells). Combined administration of the agents was not followed by increase in overall effect. Sensitivity of blood-natural killer cells in breast cancer patients to vasopressin and interleukin-2 depended upon clinical pattern, stage of tumor and treatment modality. PMID:2596062

Andrianov, I G; Dobkin, A N; Kiselev, O I; Okulov, V B; Semiglazov, V F



The Association of -475 and -631 Interleukin-2 Gene Polymorphism with Multiple Sclerosis in Iranian Patients  

PubMed Central

Objective: Multiple sclerosis (MS) is a chronic autoimmune disease due to demyelination of the central nervous system. It is believed that cytokines are involved in the pathogenesis of MS. The interleukin-2 (IL2) gene is powerful functional candidate that is involved in immune regulation and operation. In this study, for the first time, we investigated the effect of -475 A/T and -631 G/A IL2 polymorphisms on MS disease in Iranian patients. Materials and Methods: In this case-control study, 100 MS patients (mean age: 32.95 ± 6.51 years, age range: 20-42 years) selected according to McDonald criteria, and 100 ethnically, sex and age matched healthy controls (mean age: 29 ± 7.8 years, age range: 20-52 years) with no personal or family history of autoimmune diseases were studied. The restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method was applied to define different alleles and genotypes of IL2 promoter single nucleotide polymorphism -475 A/T as well as -631 G/A among individuals. ?2 was calculated and Fisher’s exact test was applied to analyze the obtained data. The value of p < 0.05 was considered significantly . Results: Evaluation of the -475 IL2 revealed that T allele and A/T genotype are present in 2% and 4% of MS patients, respectively, whereas T allele was absent in control samples. The comparison between alleles and genotypes in MS patients and healthy controls was not significant (p=0.1). For the -631 position, 1% and 2% of MS patients carried A allele and A/G heterozygote genotypes, respectively. All control samples had G allele and G/G genotype. The differences between patients and controls were not significant (p=0.4). Moreover, our results showed a very low frequency of T at -475 and A at -631 IL2 position in each of the two groups. Conclusion: Both -475 and -631 IL2 polymorphisms were higher in MS patients as compared to controls, but the frequency differences were not significant. Based on these data, it is suggested that the -475 and -631 IL2 polymorphisms as functional promoter position may be involved in IL2 expression and regulation. To find out the exact effect of the mentioned SNPs on susceptibility to MS, study on a larger sample size is suggested.

Sayad, Aida; Allameh, Abdolamir; Sayad, Arezou; Noruzinia, Mehrdad; Akbari, Mohammad Taghi; Sarzaeem, Ali; Akbar, Akbari; Haji Hoseini, Reza



Suppression of interleukin-2 production by macrophages in genetically susceptible mice infected with Leishmania major.  

PubMed Central

Spleen cells from BALB/c mice infected with 2 X 10(7) L. major promastigotes and developing progressive disease produced significantly lower levels of interleukin-2 (IL-2) in response to concanavalin A stimulation than did spleen cells from uninfected mice. In contrast, spleen cells from sublethally irradiated and infected mice, which were able to contain lesion development, produced significantly higher levels of IL-2. The increase in IL-2 production closely paralleled lesion regression. Mice protectively immunized by four intravenous injections with lethally irradiated promastigotes also produced enhanced levels of IL-2, which were sustained after challenge infection. In contrast, spleen cells from BALB/c mice given four s.c. injections of irradiated promastigotes produced high levels of IL-2 before but not after infection. These mice eventually produced levels of IL-2 indistinguishable from those of unimmunized mice with progressive disease. There is thus an inverse relation between disease progression and the ability of spleen cells to produce IL-2. Spleen cells from mice with uncontrolled disease not only produced lower levels of IL-2 but also impaired IL-2 production by normal spleen cells. The ability to inhibit IL-2 was abrogated by passing the cells through a Sephadex G-10 column, removal of plastic adherent cells, and removal of carbonyl iron-ingesting cells. Furthermore, Sephadex G-10 column-treated and plastic adherent, nonspecific esterase-positive spleen cells from mice with progressive disease were able to suppress IL-2 production by normal splenic T cells. The suppressive activity of the adherent cells was not affected by treatment with anti-Thy-1.2 antibody and complement. In contrast, adherent spleen cells from uninfected mice were devoid of such suppressor activity. The depressed IL-2 production by spleen cells from progressively infected mice could be restored to that of normal spleen cells by the addition of indomethacin to the culture. There was however, no correlation between IL-2 production and IL-1 activity in infected or immunized BALB/c mice. Thus, it appears that the suppression of IL-2 production is mediated by prostaglandins elaborated by macrophages from chronically infected mice.

Cillari, E; Liew, F Y; Lelchuk, R



Administration of R24 monoclonal antibody and low-dose interleukin 2 for malignant melanoma.  


R24 is a monoclonal antibody that recognizes the disialoganglioside GD3 expressed on the surface of malignant melanoma cells. Once bound, it can mediate destruction of these cells through both complement-mediated lysis and antibody-dependent cellular cytotoxicity. Agents such as interleukin 2 (IL-2), which can augment effector cell function and promote destruction of antibody-coated tumor cells, might produce improved antitumor responses when combined with R24. In this series, we evaluated the combination of R24 and IL-2 in a Phase 1b study in patients with metastatic melanoma. Twenty-eight patients with metastatic melanoma were entered into the protocol at two institutions. Patients received 8 weeks of IL-2 by continuous i.v. infusion at a dose (4.5 x 10(5) Amgen units/m2/day) designed to selectively expand natural killer (NK) cells. In weeks 5 and 6, patients received R24 for a total of four doses. Twenty-four h after each R24 infusion, patients received a 2-h bolus dose of IL-2 to help promote activity of NK effectors against antibody-coated melanoma targets. Additional IL-2 boluses were administered in weeks 7 and 8. Doses were escalated through two bolus doses of R24 (5 or 15 mg/m2) and two bolus doses of IL-2 (2.5 or 5.0 x 10(5) units/m2). Although one patient experienced severe capillary leak syndrome during IL-2, therapy was otherwise well tolerated. At the higher dose level of R24, two of four patients experienced transient but severe abdominal and chest discomfort, necessitating dose reduction. One patient with ocular melanoma and liver metastases had a partial response. Two additional patients had minor responses. A dramatic increase in NK cell number was noted as a result of treatment, as was augmentation of cytolytic activity against cultured NK-sensitive targets. Antibody-dependent cellular cytotoxicity against cultured melanoma cells in the presence of exogenous R24 or in the presence of serum obtained from patients following R24 infusion also increased during treatment. Our experience indicates that R24 and low-dose IL-2 can be safely combined in patients with metastatic melanoma and that this combination can promote destruction of cultured melanoma cells. The clinical activity of this combination against ocular melanoma may merit further investigation. PMID:9815532

Soiffer, R J; Chapman, P B; Murray, C; Williams, L; Unger, P; Collins, H; Houghton, A N; Ritz, J



Transient expression of interleukin 2 receptors. Consequences for T cell growth  

PubMed Central

T lymphocyte mitosis results from the interaction of interleukin 2 (IL- 2) with specific receptors that appear only after appropriate immune stimulation. To assess the potential role of IL-2 receptor levels in determining the rate and magnitude of T cell proliferation, the expression of IL-2 receptors by lectin-stimulated human peripheral blood T cells was examined and correlated with T cell growth. Using biosynthetically radiolabeled IL-2 and anti-Tac, a monoclonal antibody that blocks IL-2 receptor binding, IL-2 receptors were found to accumulate slowly and asynchronously among lectin-stimulated T cells and to precede the onset of DNA synthesis. Moreover, a critical threshold of IL-2 receptor density appeared to be required before the commitment to cell cycle progression, as analyzed quantitatively by tritiated thymidine incorporation and flow cytometric analysis of cellular DNA content. Once maximal IL-2 receptor expression occurred, continued proliferation was IL-2 concentration dependent as assessed using homogenous immunoaffinity-purified IL-2. Upon removal of the activating lectin, IL-2 receptor levels progressively declined, and, in parallel, the rate of proliferation diminished. The decay of IL-2 receptors could not be attributed to IL-2-mediated down-regulation. Instead, renewed IL-2 receptor expression was dependent upon the reintroduction of the initial activating signal. Repetitive exposure to lectin resulted in a more rapid reexpression of maximal IL-2 receptor levels, which was then followed by an accelerated resumption of proliferation. Thus, the extent of T cell proliferation after immune stimulation depends upon the interplay of the IL-2 concentration available and the density of IL-2 receptors expressed, both of which are ultimately determined by antigen/lectin stimulation. The awareness of the transience and the antigen/lectin dependence of IL-2 receptor expression, together with the capacity to monitor T cell cultures for IL-2 receptor levels, should facilitate the initiation and maintenance of cloned, antigen-specific T cells in long-term culture. In addition, these findings suggest that, in vivo, the rapidity of acquisition of maximum IL-2 receptor levels by activated T cells and the duration of IL-2 receptor expression may well direct the magnitude of T cell clonal expansion and resultant immune responses.



Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'.  


The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells. Considerable effort has been invested in using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary 'high affinity' receptor complex consisting of IL-2, IL-2R? (termed CD25), IL-2R? and IL-2R?. Naive T cells express only a low density of IL-2R? and IL-2R?, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2R? and IL-2R?. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 'superkine' (also called super-2) with increased binding affinity for IL-2R?. Crystal structures of the IL-2 superkine in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, reducing the flexibility of a helix in the IL-2R? binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy. PMID:22446627

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



Phase 1 study of stereotactic body radiotherapy and interleukin-2--tumor and immunological responses.  


Preclinical models suggest that focal high-dose radiation can make tumors more immunogenic. We performed a pilot study of stereotactic body radiation therapy (SBRT) followed by high-dose interleukin-2 (IL-2) to assess safety and tumor response rate and perform exploratory immune monitoring studies. Patients with metastatic melanoma or renal cell carcinoma (RCC) who had received no previous medical therapy for metastatic disease were eligible. Patients received one, two, or three doses of SBRT (20 Gy per fraction) with the last dose administered 3 days before starting IL-2. IL-2 (600,000 IU per kilogram by means of intravenous bolus infusion) was given every 8 hours for a maximum of 14 doses with a second cycle after a 2-week rest. Patients with regressing disease received up to six IL-2 cycles. Twelve patients were included in the intent-to-treat analysis, and 11 completed treatment per the study design. Response Evaluation Criteria in Solid Tumors criteria were used to assess overall response in nonirradiated target lesions. Eight of 12 patients (66.6%) achieved a complete (CR) or partial response (PR) (1 CR and 7 PR). Six of the patients with PR on computed tomography had a CR by positron emission tomography imaging. Five of seven (71.4%) patients with melanoma had a PR or CR, and three of five (60%) with RCC had a PR. Immune monitoring showed a statistically significantly greater frequency of proliferating CD4(+) T cells with an early activated effector memory phenotype (CD3(+)CD4(+)Ki67(+)CD25(+)FoxP3(-)CCR7(-)CD45RA(-)CD27(+)CD28(+/-)) in the peripheral blood of responding patients. SBRT and IL-2 can be administered safely. Because the response rate in patients with melanoma was significantly higher than expected on the basis of historical data, we believe that the combination and investigation of CD4(+) effector memory T cells as a predictor of response warrant further study. PMID:22674552

Seung, Steven K; Curti, Brendan D; Crittenden, Marka; Walker, Edwin; Coffey, Todd; Siebert, Janet C; Miller, William; Payne, Roxanne; Glenn, Lyn; Bageac, Alexandru; Urba, Walter J



Role of nitric oxide in interleukin 2-induced corticotropin-releasing factor release from incubated hypothalami.  

PubMed Central

Stimulation of corticotropin-releasing factor (CRF) release from the hypothalamus by interleukin 2 (IL-2) was recently demonstrated. Cytokines induce nitric oxide synthase (NOS), an enzyme that converts L-arginine into L-citrulline and nitric oxide (NO). NO is believed to be responsible for the cytotoxic action of these agents. The constitutive form of NOS occurs in neurons in the central nervous system and NO appears to play a neurotransmitter role in cerebellar and hippocampal function. We explored the probability that IL-2 and synaptic transmitters might release CRF via NO. The effects of L-arginine, the substrate for NOS, and NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, on IL-2-induced CRF release were studied using mediobasal hypothalami (MBHs) incubated in vitro in Krebs-Ringer bicarbonate buffer. L-Arginine did not alter basal and IL-2-induced CRF release after 30 min of incubation but significantly elevated both basal and IL-2-induced CRF release when MBHs were incubated 30 min longer, presumably because the endogenous substrate had been depleted after the initial 30-min incubation period. In 30-min incubations, both carbachol, an acetylcholineomimetic drug, and norepinephrine stimulated CRF release. There was an additive effect of incubation of the MBHs in the presence of carbachol (10(-7) M) and IL-2 (10(-13) M). On the other hand, coincubation of MBHs with norepinephrine (10(-6) M) and IL-2 (10(-13) M) did not produce any additive effect. Addition of NMMA, an inhibitor of NOS, at 1 or 3 x 10(-4) M completely suppressed IL-2-induced release of CRF as well as that caused by IL-2 plus carbachol. In contrast, the release of CRF induced by norepinephrine was not blocked by 3 x 10(-4) M NMMA. The data indicate that IL-2 can activate constitutive NOS leading to increased NO release, which activates CRF release. It appears that NO is also involved in the release of CRF induced by carbachol but not by norepinephrine.

Karanth, S; Lyson, K; McCann, S M



Enhanced tumor uptake of macromolecules induced by a novel vasoactive interleukin 2 immunoconjugate.  


Low uptake of monoclonal antibodies (MAbs) in cancer lesions is a significant problem in cancer therapy. Recent studies have shown that antibody uptake in tumor is controlled in large part by the tumor blood flow and the vascular permeability of the tumor endothelium. We have hypothesized that these physiological properties of tumor vessels may be altered by pretreatment with vasoactive drugs or peptides linked to tumor-specific MAbs. To test this hypothesis, two MAbs, Lym-1 directed against human malignant lymphomas and B72.3 reactive with the TAG-72 antigen expressed in solid tumors, were chemically conjugated with human recombinant interleukin 2 (IL-2). IL-2 has been used in humans to activate lymphokine-activated killer cells for the treatment of cancer but is also known to produce a generalized vascular permeability by an unknown mechanism when used systemically. Chemical conjugation of IL-2 to MAbs appears to destroy its cytokine function as shown by T-cell proliferation studies in vitro. Despite this finding, MAb/IL-2 immunoconjugates retain their ability to produce an enhanced vascular permeability when injected i.v. into nude mice bearing relevant tumor models only. Biodistribution studies using 125I-labeled tracer Lym-1 have demonstrated that the Lym-1/IL-2 immunoconjugate can increase antibody uptake in tumor by a factor of 4 in a time (2.5-h pretreatment)- and dose (30 micrograms/mouse)-dependent manner. In contrast, treatment of mice with free IL-2 and antibody showed this effect in all organs of the mouse including the tumor. Bidirectional crossover imaging studies in individual tumor-bearing nude mice showed improved uptake and decreased blood pool when the MAb/IL-2 immunoconjugates were used compared to controls. Finally, tumor blood flow and vascular permeability studies demonstrate that the physiological effect of the MAb/IL-2 is due to a reversible and specific vascular leakage at the tumor site. These studies indicate that pretreatment with this novel immunoconjugate may enhance the diagnostic and therapeutic potential of MAbs, drugs, and other macromolecules for the treatment of cancer. PMID:2021947

LeBerthon, B; Khawli, L A; Alauddin, M; Miller, G K; Charak, B S; Mazumder, A; Epstein, A L



Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial.  


We conducted a phase 1 trial of direct injection of an E1, E3-deleted adenovirus encoding interleukin-2 (AdCAIL-2) into subcutaneous deposits of melanoma or breast cancer. Twenty-three patients were injected at seven dose levels (10(7)-10(10) p.f.u). Local inflammation was observed at the site of injection in 60% of patients, but side-effects were otherwise minor. Incomplete local tumor regression occurred at the site of injection in 24% of patients, but no conventional clinical responses were seen. Circulating CD4 and CD8 counts fell significantly 24 h after injection. Post-injection biopsies demonstrated tumor necrosis and lymphocytic infiltration with the predominant tumor-infiltrating cells both CD3- and CD8-positive. Vector-derived sequences were detected in 14 of 18 biopsies examined 7 days after injection and vector-derived hIL-2 mRNA was detected in 80% of 7-day biopsies processed after injection of 10(8) p.f.u. of AdCAIL-2 or higher. While IL-2 was detectable by ELISA in tumor biopsies at 48 h, no protein was detectable in injected tumors after 7 days and no circulating IL-2 was detectable at any time-point. No Ad5E1 sequences were detected either before or after injection indicating absence of replication-competent virus or endogenous E1-like sequence; furthermore, only rare vector shedding was detected. Anti-adenovirus and neutralizing antibody titers were elevated 1 month after injection in all patients. This trial therefore confirms the safety of use of adenoviral vectors for gene delivery in humans and demonstrates successful transgene expression even in the face of pre-existing immunity to adenovirus. PMID:10435085

Stewart, A K; Lassam, N J; Quirt, I C; Bailey, D J; Rotstein, L E; Krajden, M; Dessureault, S; Gallinger, S; Cappe, D; Wan, Y; Addison, C L; Moen, R C; Gauldie, J; Graham, F L



Differential regulation of lymphokine production by distinct subunits of the T cell interleukin 2 receptor.  

PubMed Central

Most biologic responses to IL-2 have been attributed to interaction of IL-2 with a high affinity receptor which consists of a heterodimer composed of two distinct IL-2-binding proteins (IL-2R alpha/IL-2R beta). However, both low affinity IL-2R alpha (55 kD) and intermediate affinity IL-2R beta (70-75 kD) also appear to be expressed independently on the cell surface. We investigated the receptor-specific regulatory effects of IL-2 on cytokine production in unstimulated and activated T cells. T cells were activated by stimulation of the antigen receptor complex with anti-CD3 mAb. IL-2 (10(2) U/ml, 1 nM) stimulation of resting cells resulted in a fivefold increase in GM-CSF release but in only minimal IFN-gamma release. IL-2 markedly augmented mRNA expression of GM-CSF but not IFN-gamma in unstimulated T cells. IL-2R beta mAb but not IL-2R alpha mAb decreased IL-2-induced GM-CSF release and mRNA expression from unstimulated T cells. IL-2 concentrations required for GM-CSF release from resting cells suggested ligand binding to an intermediate affinity receptor. GM-CSF and IFN-gamma release from activated T cells increased four- to fivefold in response to 1 nM IL-2 and IL-2 augmented both GM-CSF and IFN-gamma mRNA. IL-2R beta mAb but not IL-2R alpha mAb reduced GM-CSF release and mRNA expression in activated T cells stimulated with 1 nM IL-2. IL-2R alpha blockade markedly decreased IL-2-induced IFN-gamma release and mRNA expression from activated cells, while IL-2R beta blockade had little effect on IFN-gamma production in activated cells. IL-2R alpha blockade altered the affinity of the receptor mediating activated cell GM-CSF release from a high affinity to an intermediate affinity state. These studies indicate an independent role for IL-2R beta in mediating GM-CSF production from T cells. They also suggest that unstimulated and activated T cells, which express distinct IL-2 receptor moieties, mediate release of separate lymphokines and that different subunits of the IL-2 receptor may play an important role in the regulation of cytokine production. Images

Burdach, S; Zessack, N; Dilloo, D; Shatsky, M; Thompson, D; Levitt, L




Microsoft Academic Search

Background. Interleukin-2 is an important regulatory cytokine of the immune system, with potent ef- fects on T cells, B cells, and natural killer cells. In vitro, interleukin-2 can induce the proliferation and differentia- tion of peripheral-blood mononuclear cells from patients infected with the human immunodeficiency virus (HIV). Methods. We treated 25 HIV-infected patients with in- terleukin-2 administered as a continuous



Therapeutic effect of a vaccinia colon oncolysate prepared with interleukin-2-gene encoded vaccinia virus studied in a syngeneic CC36 murine colon hepatic metastasis model  

Microsoft Academic Search

Vaccinia CC-36 murine colon oncolysate (VCO) prepared with interleukin-2-gene encoded recombinant vaccinia virus (IL-2VCO) was used in the treatment of a syngeneic murine colon adenocarcinoma (CC-36) hepatic metastasis to test the beneficial effect of the interleukin-2-gene encoded vaccinia virus over a control recombinant vaccinia virus in producing a vaccinia oncolysate tumor cell vaccine. Results suggest that the IL-2VCO treatment significantly

Muthukumaran Sivanandham; Stephen D. Scoggin; Nobuyuki Tanaka; Marc K. Wallack



Interleukin2, soluble interleukin-2-receptor, neopterin,l-tryptophan and ? 2 -microglobulin levels in CSF and serum of patients with relapsing-remitting or chronic-progressive multiple sclerosis  

Microsoft Academic Search

Cerebrospinal fluid (CSF) and serum levels of interleukin-2 (IL-2), soluble IL-2 receptors (sIL-2R), neopterin,l-tryptophan (l-TRP) and ß2-microglobulin ((ß2-M) were measured in 31 untreated multiple sclerosis patients in acute exacerbation and 27 normal controls. Twenty-six patients showed the relapsing-remitting type of disease (RRMS); 5 had a chronic-progressive course (CPMS). No changes in serum IL-2 and sIL-2R were found between RRMS patients

Melanie Ott; Lothar Demisch; Wolfram Engelhardt; Peter-Alexander Fischer



[Effect of interleukin-2 on apoptosis and function of human lymphocytes in in-vitro cytostatics culture].  


In vitro experiments were conducted to determine the impact of interleukin-2 (IL-2) on apoptosis and function of cytostatic-cultured lymphoid (mononuclear) cells (MNC) of peripheral blood from healthy subjects and children with cancer. Neither slight or any effect on vepesid-16 or carboplatin--cultured MNC apoptosis, nor any phytohemagglutinin--induced proliferation was found. By contrast, in carboplatin- cultured MNC from healthy subjects, IL-2 significantly potentiated their toxicity for tumor cells by producing interferon-gamma. It was concluded that IL-2 predominantly supported MNC functional activity rather than inhibited lymphoid MNC apoptosis in in vitro culture with cytostatic drugs. PMID:17037038

Potapnev, M P; Vashkevich, E P; Savitski?, V P; Belevtsev, M V; Ismail-zade, R S; Konoplia, N E



[An experimental study of mitogen-stimulated blastogenic transformation and interleukin-2 in mice after burn injury].  


In this study, mitogen-stimulated blastogenic transformation (MSBT) and interleukin 2 (IL-2) production by splenic lymphocytes in mice were measured at various time intervals after unanesthetized burn injury. The results showed that both MSBT and IL-2 production were suppressed after burn injury. There was a significant positive correlation between these two parameters. The postburn serum showed in vitro suppressive activity upon MSBT, IL-2 production and IL-2-IL-2R interaction of normal control. The results indicated that burn injury had a significant effect on lymphocytes. PMID:1446288

Liang, H P



Efficacy of repeated cycles of chemo-immunotherapy with Thymosin ?1 and interleukin-2 after intraperitoneal 5-fluorouracil delivery  

Microsoft Academic Search

We have used chemo-immunotherapy with 5-fluorouracil (5-FU), thymosin ?1 (T?1) and interleukin-2 (IL-2) to treat multiple\\u000a liver metastases from colorectal cancer induced by DHD\\/K12 cells in syngeneic BDIX rats, comparing one and two cycles of treatment,\\u000a and different treatment combinations. 5-FU was delivered loco-regionally as a continuous infusion via an intraperitoneal (i.p.)\\u000a catheter from a subcutaneously implanted mini-pump, a method

Gianfranco Silecchia; Enrico Guarino; Paola Sinibaldi-Vallebona; Pasquale Pierimarchi; Angelo Restuccia; Erasmo Spaziani; Paola Bernard; Cynthia Tuthill; Enrico Garaci; Guido Rasi



Simple purification of Escherichia coli -derived recombinant human interleukin-2 expressed with N-terminus fusion of glucagon  

Microsoft Academic Search

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed inEscherichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates\\u000a of recombinant fusion protein produced inE. coli cytoplasm were easily dissolved by simple alkaline pH shift (8?12?8). Following enterokinase cleavage, the recombinant hIL-2\\u000a was finally purified by

Hye-Soon Won; Jeewon Lee; In-Ho Kim; Young-Hoon Park



Interleukin-2 regulates the expression of the tumor suppressor Interleukin-24 in melanoma cells  

PubMed Central

Melanoma is notoriously resistant to chemotherapy, but variable responses to biotherapies, including the IFNs and IL-2, provide intriguing avenues for further study. Systemic IL-2 treatment has provided significant clinical benefit in a minority of metastatic melanoma patients, leading to long term survival in a few cases. We hypothesize that one previously unidentified mechanism of effective IL-2 therapy is through direct upregulation of the tumor suppressor IL-24 in melanoma tumor cells resulting in growth suppression. In this study five melanoma cell lines were treated with high dose recombinant human IL-2. Three (A375, WM1341, WM793) showed statistically significant increases in IL-24 protein; two (WM35, MeWo) remained negative for IL-24 message and protein. This increase was abolished by preincubating with anti-IL-2 antibody or blocking with antibodies against the IL-2 receptor chains. These IL-2 responsive melanoma cell lines expressed IL-2R? and ? mRNA. The IL-2R?? complex was functional, as measured by IL-2-induced STAT activation as well as IL-15 signaling through its shared receptor complex. IL-24 upregulation was observed in response to either IL-2 or IL-15. Cell growth was significantly decreased by treatment of IL-24 positive cells with IL-2 or IL-15, while no effect was seen in negative cells. Incubating the IL-24 inducible-cells with anti-IL-24 antibody as well as transfecting with IL-24 siRNA effectively reversed the growth suppression seen with IL-2. Thus, we have shown that one mechanism of clinically effective IL-2 therapy may be the direct action of IL-2 on a biologically distinct subset of melanoma cells leading to upregulation of the tumor suppressor IL-24.

Jen, Emily Y.; Poindexter, Nancy J.; Farnsworth, Elizabeth S.; Grimm, Elizabeth A.



New approach for in vivo detection of insulitis in type I diabetes: activated lymphocyte targeting with 123I-labelled interleukin 2.  


Insulitis is considered the histopathological hallmark of type I (insulin-dependent) diabetes. In the non-obese diabetic (NOD) mouse, diabetes has never been observed in the absence of insulitis. The in vivo detection of insulitis could be of relevance for early prediction of diabetes. As approximately 15% of islet-infiltrating lymphocytes express interleukin 2 receptors, we have labelled recombinant interleukin 2 with 123I and used this radiopharmaceutical to detect insulitis by gamma camera imaging. We studied 71 prediabetic NOD and 27 normal Balb/c mice. Labelled alpha-lactalbumin was used as the control protein. In the first set of experiments we studied the tissue distribution of radiolabelled interleukin 2 in isolated organs from animals sacrificed at different time points. Higher radioactivity was detected in the pancreas of NOD mice injected with labelled interleukin 2, as compared to NOD mice receiving labelled alpha-lactalbumin (p < 0.003 at 20 min: p < 0.001 at 40 min; p < 0.001 at 60 min) or Balb/c mice injected with labelled interleukin 2 (p < 0.05 at 40 min; p < 0.001 at 60 min). In another set of experiments, gamma camera images have been acquired after injection of 123I-labelled interleukin 2. Radioactivity in the pancreatic region of prediabetic NOD and Balb/c mice showed similar kinetics to those observed by single organ counting, with higher accumulation in the pancreatic region of NOD mice (p < 0.04 after 22-45 min in NOD mice vs Balb/c mice).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7921233

Signore, A; Chianelli, M; Ferretti, E; Toscano, A; Britton, K E; Andreani, D; Gale, E A; Pozzilli, P



hNT neurons express an immunosuppressive protein that blocks T-lymphocyte proliferation and interleukin-2 production.  


Ntera2/D1 cells had an A1 B8 Bw6 Cw7 DR3 DR52 major histocompatibility complex (MHC) genotype. Its neuronal derivative, hNT neurons, expressed A1 B8 Bw6 MHC class I molecules, but did not activate, and its hNT supernatant suppressed allogeneic mixed lymphocyte cultures (MLC) >98% (p<0.01), phytohemagglutinin (PHA)-activated T-cell proliferation >87% (p<0.01), even 48 h after stimulation, suppressed phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced T-cell proliferation >99% (p<0.001), and reduced interleukin-2 (IL-2) production (p<0.01), while maintaining T cells in a quiescent G(0)/G(1) state without lowering their viability. This immunosuppressive activity was attributed to a 40-100-kDa anionic hNT protein with an isoelectric point of 4.8. PMID:11960646

Gower, W R; Sanberg, P R; Brown, P G; Salyani, S; Pasqual, C J; McGrogan, M; Good, R A; Engelman, R W



Effects of amorphous and crystalline nickel sulfide on induction of induction of interferons-. alpha. /. beta. and -. gamma. and interleukin-2  

SciTech Connect

Pretreatment of rat spleen cells with crystalline or amorphous nickel sulfide (NiS) did not inhibit induction of interferon-{gamma} or interleukin-2 by concanavalin A. In contrast, pretreatment of murine L-929 cells with crystalline NiS inhibited severely IFN-{alpha}/{beta} induction by polyriboinosinic-polyribocytidylic acid, while amorphous NiS did not show any effect. This difference in the effects of both forms of NiS on the spleen and L-929 cells correlated with the cellular internalization of the compound; L-929 cells actively phagocytosed crystalline NiS while the uptake of amorphous NiS by the same type of cells was minimal. The uptake of both crystalline and amorphous NiS by spleen cells was very low.

Jaramillo, A.; Sonnenfeld, G. (Univ. of Louisville, KY (USA))



Differential effects of human and porcine interleukin 2 on natural killing (NK) activity of newborn piglets and adult pigs lymphocytes.  


The in vitro effects of human or porcine Interleukin 2 (IL2) on Natural Killing (NK) activity were studied with blood lymphocytes of newborn piglets and of adult pigs. Large volumes of porcine IL2 were prepared by PHA stimulation of irradiated mesenteric lymph node cells and, following purification by gel-filtration chromatography, the apparent molecular weight of IL2 was 15,000 Da. Purified human and porcine IL2 as well as recombinant human IL2 were found to increase markedly NK activity of lymphocytes derived from adult animals. However, although newborn piglets-derived lymphocytes are sensitive to crude porcine IL2 supernatants (and to Interferon alpha as previously shown) their low NK activity is unaffected by purified IL2. These data suggest therefore the existence of differences in the ontogenic development of the lymphokines responsiveness of porcine NK cells. PMID:3501262

Charley, B; Fradelizi, D



The stimulation of EL-4 cells to produce interleukin-2 and its potential use in immunocytotoxicity testing  

SciTech Connect

The ability of EL-4 thymoma cells to produce interleukin-2 (IL-2) following exposure to phorbol-12-myristate 13-acetate (PMA) and Concanavalin A (Con A) has been studied in vitro using medium containing either 10% or 1% fetal calf serum (FCS). The potent stimulatory effect of PMA on IL-2 production by EL-4 cells has been confirmed by measuring 3H-thymidine incorporation by the IL-2-dependent T cell line, CTLL-2, in the presence of conditioned medium (CM) from stimulated cultures. EL-4 cells produced several times more IL-2 when cultured in medium containing 10% FCS than when only 1% FCS was present. Added together, PMA and Con A acted synergistically in some EL-4 cell cultures. The ability of E:-4 cells to produce IL-2 was maintained after further incubation without stimulants. CM with IL-2 activity from stimulated EL-4 cells could prove useful in immunotoxicity testing.

Lasek, W.; Steer, S.; Clothier, R.; Balls, M. (Univ. of Nottingham Medical School, Queen's Medical Centre (England))



A herpesvirus saimiri membrane protein required for interleukin-2 independence forms a stable complex with p56lck.  

PubMed Central

ORF-2, a 32-kDa viral protein expressed by herpesvirus saimiri-transformed lymphocytes, is essential for transformation and is expressed on the plasma membrane of transformed cells. The current work now shows that most (approximately 80%) of ORF-2 resides in the cytoplasm, while only a small portion protrudes from the cell surface. Expressed as a glutathione S-transferase fusion protein, ORF-2 was found to interact with a 56-kDa cellular protein in untransformed, herpesvirus saimiri-transformed, and Jurkat lymphocytes. Microsequencing proved that this protein is the lymphocyte-specific tyrosine protein kinase p56lck. Two regions of ORF-2 were found to be required for p56lck interaction. Current evidence suggests that the interaction of ORF-2 with p56lck plays a key role in the specific transformation of T lymphocytes to an interleukin-2-independent phenotype.

Lund, T; Medveczky, M M; Neame, P J; Medveczky, P G



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


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

Tyre, Charley Cowan; Quan, Walter



Reduction of the acetylcholine-induced K+ current in identified Aplysia neurons by human interleukin-1 and interleukin-2.  


1. Effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with voltage-clamp and pressure ejection techniques. 2. Bath-applied rhIL-1 and rhIL-2 (10-40 U/ml) reduced the ACh-induced current in the neurons without affecting the resting membrane conductance and holding current. 3. The suppressing effects of these cytokines on the current were completely reversible. 4. Heat-inactivated rhIL-1 and rhIL-2 were without effect. 5. These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the ACh-induced response in the nervous system. PMID:1468114

Sawada, M; Hara, N; Maeno, T



Murine T-lymphocyte proliferation induced by interleukin 2 correlates with a transient increase in p56lck kinase activity and the tyrosine phosphorylation of a 97-kDa protein.  

PubMed Central

The addition of recombinant interleukin 2 (rIL-2) to anti-CD3-activated murine G0 phase T cells results in an increased level of tyrosine phosphorylation of a single 97-kDa protein. The degree of tyrosine phosphorylation paralleled the amount of rIL-2 added and correlated with the extent of DNA synthesis. IL-2 treatment resulted in a transient increase in p56lck kinase activity without detectable modification of its level of tyrosine phosphorylation and gel mobility. When G0 T cells were activated by phorbol dibutyrate in the absence of IL-2, the high-affinity IL-2 receptor (IL-2R) expressed failed to induce a proliferative signal, and neither the tyrosine phosphorylation of the 97-kDa protein nor the transient increase in p56lck kinase activity was detected. Northern analysis of the total RNA extracted from these cells showed the accumulation of IL-2R alpha chain-specific mRNA but neither c-myc nor cdc2 mRNA was expressed. The addition of 100 nM rIL-2 to T cells activated by phorbol dibutyrate was able to induce a proliferative response, and under these conditions tyrosine phosphorylation of the 97-kDa protein, the transient increase in p56lck kinase activity, and specific mRNA for IL-2R alpha chain, c-myc, and cdc2 were detected. Unstimulated G0 T cells responded to 100 nM rIL-2 in the same manner as phorbol dibutyrate-activated cells. Irrespective of the signal-transducing structures involved, the IL-2-induced proliferative response closely correlates with an increase in p56lck kinase activity along with the tyrosine phosphorylation of a 97-kDa protein. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

Kim, Y H; Buchholz, M J; Nordin, A A



Soluble interleukin 2 receptors are released from the cell surface of normal murine B lymphocytes stimulated with interleukin 5.  

PubMed Central

Murine T and B lymphocytes can be induced to release soluble interleukin 2 receptors (IL2Rs). This receptor is believed to be a truncated form of the 55-kDa chain of the cell-membrane-associated receptor. It has been speculated that this receptor may play an important immunoregulatory role by binding to interleukin 2 (IL-2). We report here that interleukin 5 can induce normal murine B cells to release soluble IL2Rs. This extends our finding that interleukin 5 similarly can induce murine B cells to express functional cell-surface-associated IL2Rs. Two possible mechanisms of release of soluble IL2Rs have been suggested. Soluble IL2R could be synthesized as a secretory form of the receptor lacking the transmembrane domain or by cleavage of the extracellular domain of the cell-surface-associated IL2R at the cell surface. To investigate which mechanism was operative, we radioiodinated the cell surface of normal murine splenocytes that had been cultured for 1 day with Con A to stimulate the expression of cell-surface-associated IL2Rs and the release of soluble IL2Rs. Under the conditions used, radiolabeling of internal proteins was not apparent. Labeled cells were then recultured with Con A, conditioned medium was taken from replicate cultures at various times after radioiodination, and the specific radioactivity of released soluble IL2Rs was determined by ELISA and RIA. We demonstrate that the specific radioactivity and the kinetics of change of the specific radioactivity are consistent with the hypothesis that the soluble IL2Rs are derived from the cell-surface-associated IL2Rs rather than being released in a secretory form. Images

Loughnan, M S; Sanderson, C J; Nossal, G J



Interleukin-2 enhances the cytotoxic activity of circulating natural killer cells in patients with chronic heart failure.  


Our objective was to investigate the effect of interleukin-2 (IL-2) on the cytotoxic activity of natural killer (NK) cells in patients with chronic heart failure (CHF). Natural killer cells were isolated from 48 patients with CHF and 30 healthy subjects. The cytotoxic activities of the NK cells were assessed with the thiazolyl blue tetrazolium bromide (MTT) approach. Interleukin-2 (20 ng/ml) was added to the cell culture to stimulate the cytotoxicity of the NK cells. The cell number in the New York Heart Association (NYHA) class II, III, and IV was 27.9 +/- 2.5, 21.2 +/- 2.7, and 16.8 +/- 2.6 cells/microl, respectively, which was significantly lower than in the control group (31.2 +/- 3.6 cells/microl, all P < 0.01). The cytotoxic activities in NYHA II, III, and IV groups were 28.6% +/- 3.2%, 16.0% +/- 2.2%, and 12.1% +/- 2.9%, respectively, which was lower than in the control group (41.0% +/- 4.0%, all P < 0.01). Univariate analysis showed a close correlation between the NK cell cytotoxicity and the left ventricular ejection fraction (r = 0.949, P < 0.001). After in vitro treatment with IL-2, there was a significant increase in the cytotoxic activity of the NK cells in the control and the three heart failure groups (all P < 0.01). There is a significant reduction in the number and the cytotoxic activity of circulating NK cells in patients with CHF. The degree of NK cell deficiency is closely related to the severity of left ventricular dysfunction. In vitro treatment with IL-2 improves the cytotoxic activity of NK cell from the CHF patients. PMID:19626401

Yao, Heng-Chen; Liu, Shu-Qin; Yu, Ke; Zhou, Min; Wang, Le-Xin



Elf1 and Stat5 Bind to a Critical Element in a New Enhancer of the Human Interleukin2 Receptor aGene  

Microsoft Academic Search

The interleukin 2 receptor a-chain (IL-2Ra) gene is a key regulator of lymphocyte proliferation. IL-2Rais rapidlyandpotentlyinducedinTcellsinresponsetomitogenicstimuli.Interleukin2(IL-2)stimulatesIL-2Ra transcription, thereby amplifying expression of its own high-affinity receptor. IL-2Ratranscription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides 2276 and 2244, which contains NF-kB and SRE\\/CArG motifs. PRRII is




Characterization of Cytokine and iNOS mRNA Expression in situ During the Course of Experimental Autoimmune Myocarditis in Rats  

Microsoft Academic Search

Ribonuclease protection assay was used to demonstrate mRNA expression of several cytokines as well as inducible NO synthase (iNOS), constitutive endothelial NO synthase (cNOS) and perforin in the myocardium during the course of experimental autoimmune myocarditis (EAM) in rats. Interleukin 2 (IL-2) appeared in the initial inflammatory phase (day 14), subsided in the maximum inflammatory phase (day 19) and disappeared

Yuji Okura; Tadashi Yamamoto; Shin Goto; Takayuki Inomata; Satoru Hirono; Haruo Hanawa; Lili Feng; Curtis B. Wilson; Itaru Kihara; Tohru Izumi; Akira Shibata; Yoshifusa Aizawa; Shuhji Seki; Toru Abo



Peripheral Blood and Bone Marrow Immunophenotypicand Functional Modifications Induced in Acute Leukemia Patients Treated with Interleukin 2: Evidence of in VivoLymphokine Activated Killer Cell Generation1  

Microsoft Academic Search

The effect of treatment with interleukin 2 (112) on the phenotypic and functional immune system of acute leukemia patients was investigated. Fifteen acute myeloid leukemia and acute lymphoid leukemia patients with evidence of persistent disease were further subdivided into two groups according to the percentage of bone marrow (BM) blasts: group a had 6-15% blasts and group b had 30-65%.

Robert Foa; Anna Guarini; Anna Gillio Tos; Silvia Cardona; Maria Teresa Fierro; Giovanna Meloni; Silvia Tosti; Franco Mandelli; Felice Gavosto


Phase II Study of Interferon-gamma Versus Interleukin2 and Interferon-alpha 2b in Metastatic Renal Cell Carcinoma  

Microsoft Academic Search

PurposeIn a randomized phase II study we evaluated response, survival and side effects of low dose recombinant interferon-gamma in 30 patients (group 1) versus recombinant interleukin-2 and interferon-alpha2b in 30 (group 2) with metastatic renal cell carcinoma.

Gerd Lummen; Mark Goepel; Stefan Mollhoff; Axel Hinke; Thomas Otto; Herbert Rubben



Expression of chicken interleukin-2 by turkey herpesvirus increases the immune response against Marek's disease virus but fails to increase protection against virulent challenge  

Microsoft Academic Search

As Marek's disease virus continues to evolve towards greater virulence, more efficacious vaccines will be required in the future. We expressed chicken interleukin-2 (IL-2) from a turkey herpesvirus (HVT) in an attempt to increase the efficacy of HVT as a vaccine against Marek's disease. The recombinant IL-2\\/HVT was safe for in ovo vaccination, although it replicated less in the birds

I. Tarpey; P. J. Davis; P. Sondermeijer; C. van Geffen; I. Verstegen; V. E. J. C. Schijns; Jill Kolodsick; R. Sundick



Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.  

PubMed Central

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation. Images Fig. 1 Fig. 2 Fig. 3

Fujii, H; Nakagawa, Y; Schindler, U; Kawahara, A; Mori, H; Gouilleux, F; Groner, B; Ihle, J N; Minami, Y; Miyazaki, T



Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: D,L-. alpha. -difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2  

SciTech Connect

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4-8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4-8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE{sub 2} or cyclosporine A. The glycolytic activity, general protein synthesis (({sup 3}H)leucine incorporation), and the cell cycle progression from G{sub 2}/M to G{sub 1} are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine.

Mihm, S.; Risso, A.; Stoehr, M.; Oberdorfer, F.; Droege, W. (German Cancer Research Center, Heidelberg (West Germany))



Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells.  

PubMed Central

A cDNA sequence coding for mouse interleukin 2 (IL-2) has been cloned from a cDNA library prepared from mRNA derived from a concanavalin A-activated mouse T-cell clone. The library was constructed by using the pcD vector system, which permits the expression of cDNA inserts in mammalian cells. Screening of the library was performed by transfecting COS-7 monkey cells with pools of cDNA clones in order to express the products encoded by full-length cDNA inserts. By assaying the supernatant fluid, IL-2 cDNA clones that express T-cell growth-factor (TCGF) activity were identified. The DNA sequence codes for a polypeptide of 169 amino acid residues including a putative signal peptide. The mouse IL-2 amino acid sequence deduced from the nucleotide sequence of its cDNA shares extensive homology with the human IL-2 amino acid sequence reported previously. These results demonstrate that identification of full-length cDNA clones for many lymphokines may be achieved entirely on the basis of detection of the functional polypeptides in mammalian cells. Images

Yokota, T; Arai, N; Lee, F; Rennick, D; Mosmann, T; Arai, K



Turkey and chicken interleukin-2 cross-react in in vitro proliferation assays despite limited amino acid sequence identity.  


We cloned the cDNA of turkey interleukin-2 (IL-2), initially using oligonucleotide primers based on the sequence of the chicken IL-2 gene. Compared with the only other cytokines available for comparison, the interferons (IFN), the coding regions of the turkey and chicken IL-2 genes are much less conserved (86.24% nucleotide identical and 69.93% amino acid identical). The lack of nucleotide conservation was spread across the entire length of the coding region. In comparison, the promoters of the two avian IL-2 genes shared a high degree of identity (95.71% identical over 380 nucleotides). Phylogenetic analysis shows that turkey and chicken IL-2 have diverged to a greater extent than IL-2 from closely related mammalian species. Surprisingly, considering the low level of amino acid identity, including residues known to be important in binding the IL-2 receptor in mammalian species, both turkey and chicken IL-2 cross-react in functional assays. PMID:10714551

Lawson, S; Rothwell, L; Kaiser, P



Antitumor activity of tumor-targeted RNA replicase-based plasmid that expresses interleukin-2 in a murine melanoma model.  


Double-stranded RNA (dsRNA) has multiple antitumor mechanisms that may be used to control tumor growth. Previously we have shown that treatment of solid tumors with a plasmid that encodes Sindbis viral RNA replicase complex, pSIN-?, significantly inhibited the growth of tumors in mice. In the present study, we evaluated the feasibility of further improving the antitumor activity of the pSIN-? plasmid by incorporating interleukin-2 (IL2) gene into the plasmid. The resultant pSIN-IL2 plasmid was delivered to mouse melanoma cells that overexpress the sigma receptor. Here we report that the pSIN-IL2 plasmid was more effective at controlling the growth of B16 melanoma in mice when complexed with sigma receptor-targeted liposomes than with the untargeted liposomes. Importantly, the pSIN-IL2 plasmid was more effective than pSIN-? plasmid at controlling the growth of B16 melanoma in mice, and B16 tumor-bearing mice that were treated with pSIN-IL2 had an elevated number of activated CD4(+), CD8(+), and natural killer cells, as compared to those treated with pSIN-?. The RNA replicase-based, IL2-expressing plasmid may have applications in melanoma gene therapy. PMID:23641783

Rodriguez, B Leticia; Blando, Jorge M; Lansakara-P, Dharmika S P; Kiguchi, Yuriko; DiGiovanni, John; Cui, Zhengrong



Biochemical and biological characterization of lymphocyte regulatory molecules. V. Identification of an interleukin 2-producing human leukemia T cell line  

PubMed Central

To isolate a stable tumor cell line capable of producing human interleukin 2 (IL-2; formerly referred to as T cell growth factor), 16 human T and B leukemia cell lines were screened for constitutive and mitogen-stimulated IL-2 production. We found that the T cell leukemia line designated Jurkat-FHCRC produced > 200 U/ml of IL-2 activity after a 24-h stimulation with T cell mitogens. Peak mitogen-induced IL-2 activity was found in supernates harvested from 24-h Jurkat-FHCRC cell cultures stimulated with either 1% phytohemagglutinin or 20 microgram/ml concanavalin A. Addition of the fatty acid derivative phorbol myristate acetate to mitogen-stimulated cultures increased Jurkat-FHCRC IL-2 production to concentrations > 400 U/ml. IL-2 activity observed in such cases represented between 100--300 times that produced in conventional cultures of mitogen- or alloantigen-stimulated normal human peripheral blood or splenic lymphocytes. Jurkat-FHCRC- derived conditioned medium demonstrated equal capacity to promote the sustained in vitro proliferation of either murine or human activated T cell lines confirming the ability of Jurkat-FHCRC cells to produce human IL-2. These studies identify a new source of human IL-2 and establish a valuable reagent for the isolation and further molecular characterization of this immunoregulatory molecule.



Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity.  


The cDNAs encoding bovine and porcine interleukin-2 (IL-2) have been expressed using the baculovirus Autographa californica nuclear polyhedrosis virus as a vector in insect cells. Insect cells infected with recombinant viruses secreted bovine and porcine IL-2 into the culture medium, with biological activities for maintaining the proliferation of homologous cells. When the activities of these two IL-2 proteins and commercially available human IL-2 were tested on heterologous cells differences were found. Recombinant bovine (rb)IL-2 only supported the growth of bovine lymphocytes and was not active on human, mouse or porcine lymphocytes. Recombinant porcine (rp)IL-2 and recombinant human (rh)IL-2 supported the proliferation of human, bovine, porcine and murine cells. However, the proliferative response of human lymphocytes to rpIL-2 was only 50% of that seen with rhIL-2. Sequence differences at the predicted p55 and p75 contact binding sites may explain this. PMID:8042283

Collins, R A; Tayton, H K; Gelder, K I; Britton, P; Oldham, G



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

PubMed Central

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

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



Effects of two basidiomycete species on interleukin 1 and interleukin 2 production by macrophage and T cell lines.  


Two basidiomycete species, Lentinus edodes mycelia (LEM) and Cordyceps sinensis (CS) were examined for induction of cytokines in murine macrophage cell line R309 (R309) and T cell line LBRM-33 1A5 (1A5). When lipopolysaccharide (LPS)-activated R309 were exposed to the extracts of basidiomycetes, R309 induced significant levels of interleukin 1 (IL-1). Interleukin 2 (IL-2) induction was recognized in 1A5 cultures in the presence of IL-1 and phytohemagglutinin (PHA). However, no enhancement of IL-2 production by these basidiomycetes was discerned in 1A5 cultures with IL-1 and PHA, i.e., direct action of basidiomycetes was not found on IL-2 production of 1A5. PHA-stimulated 1A5 exposed to basidiomycetes induced IL-2 without IL-1 when co-cultured with LPS-activated R309 as a source of IL-1. Effects of basidiomycetes on IL-2 production in 1A5 seemed to be caused through their action on macrophages. The induction of IL-2, Th1 type cytokine in T lymphocyte, is a significant finding since basidiomycetes, taken as a dietary supplement for immuno-suppressed patients, especially cancer patients, would be helpful in improving their immune activity against cancer. PMID:19913939

Kawanishi, Takashi; Ikeda-Dantsuji, Yurika; Nagayama, Ariaki



New ultradeformable drug carriers for potential transdermal application of interleukin-2 and interferon-alpha: theoretic and practical aspects.  


Transfersomes (TFs) are highly deformable hydrophilic lipid vesicles that are able to penetrate the skin barrier spontaneously because of their characteristics. Transfersomes are able to transport noninvasively low- and high-molecular-weight molecules into the body. We describe the formulation and several biologic characteristics of interleukin-2 (IL-2)- and interferon-alpha (IFNalpha )-containing TFs. TFs contain natural phosphatidylcholine and sodium cholate. Recombinant human IL-2 and human hybrid IFNalpha were added to TFs and incubated for 24 hours at 4 degrees C. Immunotransfersomes were isolated from free IL-2 and IFNalpha by filtration (Centrisart, Sartorius). The biologic activity of immunotransfersomes was measured by a cytotoxic lymphoid line assay for IL-2 and by an A549-encephalomyocarditis virus assay for IFN; concentrations of proteins were determined by the enzyme-linked immunosorbent assay (ELISA). It was possible to incorporate a large amount of IL-2 and IFN in TFs (75-80%), and the incorporated IL-2, and IFN were biologically active. The increased lipid/protein ratio (90.9/1.0) led to a growing probability of association. We were thus able to show that IL-2 and IFN are trapped by transfersomes in a biologically active form and in sufficient concentrations for immunotherapy. In upcoming experiments these IL-2- and IFN-containing TFs will be used for a transdermal approach in the murine RENCA cell line model. PMID:11071458

Hofer, C; Hartung, R; Göbel, R; Deering, P; Lehmer, A; Breul, J



Nef Enhances Human Immunodeficiency Virus Replication and Responsiveness to Interleukin-2 in Human Lymphoid Tissue Ex Vivo  

PubMed Central

The nef gene is important for the pathogenicity associated with simian immunodeficiency virus infection in rhesus monkeys and with human immunodeficiency virus type 1 (HIV-1) infection in humans. The mechanisms by which nef contributes to pathogenesis in vivo remain unclear. We investigated the contribution of nef to HIV-1 replication in human lymphoid tissue ex vivo by studying infection with parental HIV-1 strain NL4-3 and with a nef mutant (?nefNL4-3). In human tonsillar histocultures, NL4-3 replicated to higher levels than ?nefNL4-3 did. Increased virus production with NL4-3 infection was associated with increased numbers of productively infected cells and greater loss of CD4+ T cells over time. While the numbers of productively infected T cells were increased in the presence of nef, the levels of viral expression and production per infected T cell were similar whether the nef gene was present or not. Exogenous interleukin-2 (IL-2) increased HIV-1 production in NL4-3-infected tissue in a dose-dependent manner. In contrast, ?nefNL4-3 production was enhanced only marginally by IL-2. Thus, Nef can facilitate HIV-1 replication in human lymphoid tissue ex vivo by increasing the numbers of productively infected cells and by increasing the responsiveness to IL-2 stimulation.

Glushakova, Svetlana; Grivel, Jean-Charles; Suryanarayana, Kalachar; Meylan, Pascal; Lifson, Jeffrey D.; Desrosiers, Ronald; Margolis, Leonid



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

NASA Astrophysics Data System (ADS)

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.

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



Coumestrol, bisphenol-A, DDT, and TCDD modulation of interleukin-2 expression in activated CD+4 Jurkat T cells.  


Endogenous estrogens are known to modulate several components of immune response, including interleukin-2 (IL-2) production. IL-2 is a cytokine that plays an important role in adaptive immune responses. These responses may be modulated by xenoestrogens such as coumestrol, bisphenol A (BPA), DDT, and TCDD. In this research, we examined the effects and potential mechanisms of action of these estrogenic compounds on IL-2 production in activated CD4+ Jurkat T cells. IL-2 production was analyzed by ELISA and Western Blot. At the transcriptional level, protein expression was examined by RT-PCR. Coumestrol, DDT and TCDD (but not BPA) significantly suppressed IL-2 production in activated CD4+ Jurkat T cells, at the transcriptional and translational levels. The transcriptional suppression of IL-2 was associated with decreased protein levels of NF-kappabeta, an important IL-2 positive transcription factor, without affecting the expression of Ikappa-Balpha protein expression, an important inhibitor of NF-kappabeta nuclear translocation. Although the direct mechanisms of xenoestrogens modulation of the immune system remain to be elucidated, coumestrol-, DDT- and TCDD-induced suppression of IL-2 may have ramifications for our understanding of the impact of xenoestrogens on health and disease. PMID:16696175

Ndebele, Kenneth; Tchounwou, Paul B; McMurray, Robert W



Capacity to produce interleukin 2 is impaired in haemophilia in the absence and presence of HIV 1 infection.  


Capacity to produce interleukin-2 (IL-2) was measured in haemophiliacs from a well-defined treated cohort. Patients were selected on the basis of HIV-1 antibody status, mean annual dose of clotting factor and liver disease severity. T-cell subsets and peripheral blood mononuclear cell proliferation to Mycobacterium tuberculosis purified protein derivative (PPD) were also measured. Haemophiliacs had reduced IL-2 production, independent of HIV-1 antibody status, mean annual dose of clotting factor concentrate used and liver disease severity. In HIV-1 antibody positive patients reduced levels correlated with PPD proliferative responses (r = 0.6, P = 0.04) and CD8 + ve (r = 0.5, P = 0.05) but not CD4 + ve cell numbers (r = 0.3, P = 0.2). No such correlations were seen in HIV-1 antibody negative patients. Reduced IL-2 production in HIV-1 antibody negative haemophiliacs was due to a qualitative defect. In HIV-1 positive patients a qualitative defect in T lymphocytes that selectively proliferate in response to PPD was observed. CD4 + ve cell numbers were reduced in HIV-1 positive patients. PMID:2121264

Madhok, R; Gracie, J A; Smith, J; Lowe, G D; Forbes, C D



Induction of graft versus leukemia effect in bone marrow transplantation: dosage and time schedule dependency of interleukin 2 therapy.  


The present work is a continuation of our studies to improve the graft versus leukemia (GVL) effect in autologous bone marrow transplantation. We have recently shown that the GVL effect of bone marrow transplantation (BMT) with interleukin 2 (IL-2)-activated bone marrow (ABM) followed by IL-2 therapy immediately after BMT is superior to the GVL effect of BMT with fresh, syngeneic bone marrow, with or without IL-2 therapy, in mice with acute myeloid leukemia. The present studies show that institution of IL-2 treatment 1, 2, or 3 weeks after BMT with ABM resulted in shortening of survival and fall in cure rate as compared to IL-2 therapy instituted immediately after BMT with ABM. Increasing the dose of IL-2 did not improve results. However, reducing the frequency of IL-2 administration to once a day instead of twice a day affected the results adversely. Commencing IL-2 therapy 1, 2, or 3 weeks after BMT with fresh, syngeneic bone marrow did not improve the GVL effect as compared to IL-2 therapy started immediately after BMT with fresh, syngeneic bone marrow. Cryopreserved bone marrow was effectively activated with IL-2 and used successfully for BMT after thawing. The animals cured of leukemia by BMT with ABM and and IL-2 therapy were not resistant to leukemia and died when reinfused with leukemic cells. Our findings suggest that for optimum GVL effect, activation of bone marrow is necessary and IL-2 therapy should be started immediately after BMT with ABM. PMID:2009520

Charak, B S; Brynes, R K; Katsuda, S; Groshen, S; Chen, S C; Mazumder, A



Interleukin-2 (IL-2) and IL-2-activated bone marrow in transplantation: evaluation from a clinical perspective.  


Incubation of bone marrow (BM) with interleukin-2 (IL-2) in vitro results in generation of killer cells providing a tool for enhancing the graft-versus-tumor effect in transplantation. We have evaluated the influence of IL-2 on the progenitor cell activity (PCA), homing pattern of BM and hemopoiesis in a syngeneic bone marrow transplantation (BMT) model in mice. The PCA index and homing pattern of BM activated with IL-2 in vitro for 24 h (ABM) were similar to those of fresh bone marrow (FBM). In vitro culture of BM for more than 1 day resulted in progressive decline in its PCA index; this was not related to the presence or absence of IL-2 in the culture medium. Toxicity of IL-2 was related to the dose and not the time of institution of IL-2 therapy after BMT. Maximum tolerated dose of IL-2 instituted immediately after BMT was 10 times higher than the dose in a non-BMT setting. The pattern of marrow reconstitution following BMT with ABM was comparable to that with FBM. This study shows that BMT with BM activated with IL-2 for 24 h results in normal hemopoiesis, and IL-2 therapy instituted immediately after BMT with ABM does not cause additional toxicity. PMID:1628133

Charak, B S; Agah, R; Brynes, R K; Chogyoji, M; Groshen, S; Chen, S C; Mazumder, A



Adoptive transfer of anti-cytomegalovirus effect of interleukin-2-activated bone marrow: potential application in transplantation.  


This work is a continuation of our studies that showed that interleukin-2 (IL-2)-activated murine bone marrow (ABM) cells have potent cytotoxic potential against murine cytomegalovirus (MCMV)-infected targets in vitro, without loss of reconstitutive ability in vivo. Our data show that ABM cells lyse the MCMV-infected cells in vitro, at both acute and chronic stages of infection; this lysis is specific for the MCMV-infected cells. ABM cells supplemented with IL-2 therapy virtually eradicated the viral infection and prolonged the survival of MCMV-infected Balb/c mice, whether or not they were immunocompromised by irradiation (P less than .001 in both situations). Efficacy of ABM cells alone or IL-2 alone was less than the combination of ABM cells and IL-2. The efficacy of combination treatment with ABM cells and IL-2 in improving the survival of MCMV-infected mice was comparable, whether used in a preventive or a therapeutic setting. Therapy with ABM plus IL-2 also prevented the reactivation of chronic MCMV infection after irradiation. Preliminary findings indicate that Thy-1+ and asialo GM1+ cells limited the MCMV proliferation by approximately 30% and 80%, respectively, while BM macrophages limited the proliferation of MCMV by 100%. These results suggest that BM transplantation (BMT) with ABM cells followed by IL-2 therapy may constitute a novel strategy to improve the host resistance against cytomegalovirus infection after BMT. PMID:1650263

Agah, R; Charak, B S; Chen, V; Mazumder, A



Regulatory dysfunction of the interleukin-2 receptor during HIV infection and the impact of triple combination therapy  

PubMed Central

The interleukin-2 (IL-2)/IL-2 receptor (IL-2R) system is the main regulatory determinant of T cell reactivity. Although it is well known that IL-2 secretion is impaired during HIV infection, up to now IL-2R expression has not been extensively studied in HIV-infected patients despite the use of IL-2 in clinical therapy trials. We show here that IL-2R expression in HIV patients with high viral load (group 1 in the study) is greatly enhanced on B lymphocytes, CD8 T lymphocytes, and monocytes, but not on CD4 T lymphocytes, compared with noninfected individuals. Paradoxically, this modified IL-2R expression does not lead to increased IL-2 responsiveness, except for B lymphocytes. In patients receiving triple combination therapy (TCT, two reverse transcriptase inhibitors and one protease inhibitor) that has triggered a drastic reduction in plasma viral load and an increase in CD4 counts (group 2 patients), IL-2R expression is significantly lower than in group 1 patients. Moreover, cells involved in cellular immunity and CD4 T lymphocytes have the capacity to respond to IL-2 after TCT. These results allow us to anticipate a beneficial role of IL-2 immunotherapy in combination with TCT.

David, Denis; Bani, Lynda; Moreau, Jean-Louis; Treilhou, Marie-Pierre; Nakarai, Takayuki; Joussemet, Marcel; Ritz, Jerome; Dupont, Bertrand; Pialoux, Gilles; Theze, Jacques



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


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

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



Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells.  

PubMed Central

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms. Images Figure 4

Hassan, J; Rainsford, E; Reen, D J



Dietary curcumin and limonin suppress CD4+ T-cell proliferation and interleukin-2 production in mice.  


Phytochemicals may reduce chronic inflammation and cancer risk in part by modulating T-cell nuclear factor-kappaB (NF-kappaB) activation. Therefore, we examined the effects of curcumin (Cur) and limonin (Lim) feeding on NF-kappaB-dependent CD4(+) T-cell proliferation. DO11.10 transgenic mice (n = 5-7) were fed diets containing 1% Cur or 0.02% Lim combined with either (n-6) PUFA [5% corn oil (CO)] or (n-3) PUFA [4% fish oil+1% corn oil (FO)] for 2 wk, followed by splenic CD4(+) T-cell isolation and stimulation with ovalbumin peptide 323-339 (OVA) and antigen-presenting cells from mice fed a conventional nonpurified rodent diet. Both Cur and Lim diets suppressed (P < 0.05) NF-kappaB p65 nuclear translocation in activated CD4(+) T-cells. In contrast, activator protein-1 (c-Jun) and nuclear factor of activated T-cells c1 were not affected compared with the CO control diet (no Cur or Lim). CD4(+) T-cell proliferation in response to either mitogenic anti-CD3/28 monoclonal antibodies (mAb) or antigenic stimulation by OVA was also suppressed (P < 0.05) by Cur as assessed by carboxyfluorescein succinimidyl ester staining. In contrast, interleukin-2 production was not directly associated with NF-kappaB status. Interestingly, dietary combination with FO enhanced the suppressive effects (P < 0.05) of Cur or Lim with respect to CD4(+) T-cell proliferation in response to anti-CD3/28 mAb. These results suggest that combination chemotherapy (FO+Cur or Lim) may favorably modulate CD4(+) T-cell-mediated inflammation. PMID:19321585

Kim, Wooki; Fan, Yang-Yi; Smith, Roger; Patil, Bhimanagouda; Jayaprakasha, Guddadarangavvanahally K; McMurray, David N; Chapkin, Robert S



Interactions between interleukin-2-activated lymphocytes and vascular endothelium: binding to and migration across specialized and non-specialized endothelia.  

PubMed Central

A prerequisite for the successful immunotherapy of solid tumours with interleukin-2 (IL-2)-activated lymphocytes is their ability to home to the tumour tissue. Lymphocyte homing is a complex process which is known to involve at least two independently regulated events: adhesion to the luminal surface of vascular endothelium and the subsequent transendothelial migration of lymphocytes. In this study we have used an in vitro model of lymphocyte homing which employs specialized high endothelium to ask whether IL-2-activated lymphocytes are able to migrate across vascular endothelium in order to leave the blood vessel. Both the adhesion of IL-2-activated cells and their migration across monolayers of cultured high endothelial cells (HEC) were increased in comparison with non-activated lymphocytes. The adhesion of IL-2-activated lymphocytes was mediated by lymphocyte function-associated antigen-1 (LFA-1) and a very late activation antigen-4 (VLA-4)-related pathway. LFA-1-dependent adhesion was mediated by ligands on HEC other than the intercellular adhesion molecule-1 (ICAM-1) and the VLA-4-related pathway was mediated by ligands other than the CS1 domain of fibronectin. HEC-adherent lymphocytes were enriched in natural killer (NK) cells and CD8+ T cells which are known to be the tumour-cytotoxic cells in IL-2-activated lymphocytes. However, there was no evidence of cytotoxicity towards the endothelial layer using a syngeneic model. The interaction of IL-2-activated lymphocytes and endothelial cells was not specific for high endothelium since equal numbers of activated lymphocytes bound to and migrated across aortic endothelium. The inability of IL-2-activated lymphocytes to discriminate between high endothelium and non-specialized 'flat' endothelium could be responsible for the widespread dissemination of the cells throughout the body following their adoptive transfer and the unwanted side-effects at non-involved sites. Images Figure 2

Pankonin, G; Reipert, B; Ager, A



Interleukin 2 regulates Raf-1 kinase activity through a tyrosine phosphorylation-dependent mechanism in a T-cell line.  

PubMed Central

Previously we found that interleukin 2 (IL-2) induces tyrosine phosphorylation and activation of the serine/threonine-specific kinase encoded by the raf-1 protooncogene in a T-cell line, CTLL-2. Here we extended these findings by exploring the effects of selective removal of phosphate from tyrosines in p72-74-Raf-1 kinase that had been immunoprecipitated from IL-2-stimulated CTLL-2 cells. Treatment in vitro of IL-2-activated Raf-1 with the tyrosine-specific phosphatases CD45 and TCPTP (formerly called T-cell protein tyrosine phosphatase) reduced Raf kinase activity to nearly baseline levels. This effect was completely inhibited by the phosphatase inhibitor sodium orthovanadate. In contrast, treatment of Raf-1 with a serine/threonine-specific phosphatase, protein phosphatase 1 (PP-1), resulted in a more modest decrease in Raf in vitro kinase activity, and this effect was prevented by okadaic acid. Two-dimensional phosphoamino acid analysis confirmed the selective removal of phosphate from tyrosine by CD45 and from serine and threonine by PP-1. The immunoreactivity of p72-74-Raf-1 with anti-phosphotyrosine antibodies was also completely abolished by treatment with CD45 in the absence but not in the presence of sodium orthovanadate. These findings provide evidence that the IL-2-stimulated phosphorylation of Raf-1 on tyrosines plays an important role in upregulating the activity of this serine/threonine-specific kinase in CTLL-2 cells and, as such, provides a model system for studying the transfer of growth factor-initiated signals from protein tyrosine kinases to serine/threonine-specific kinases. Images Fig. 1 Fig. 2 Fig. 3

Turner, B C; Tonks, N K; Rapp, U R; Reed, J C



Porcine interleukin-2 gene encapsulated in chitosan nanoparticles enhances immune response of mice to piglet paratyphoid vaccine.  


Interleukin-2 (IL-2) is vital to elicit and amplify the cellular and humoral immune responses to foreign antigens, which is extensively utilized in the control of infectious disease and treatment of various cancers. Porcine and murine IL-2 genes were, respectively, subcloned into VR1020, designated as VPIL-2 and VMIL-2, and then encapsulated in chitosan nanoparticles (CNP) prepared by ionic linkage. The BALB/c mice were intramuscularly co-administrated with chitosan-IL-2 nanoparticles (CNP-IL2) and paratyphoid vaccine to test the adjuvant effect of CNP-IL2. On day 35, the immunized mice were orally challenged with virulent Salmonella. The content of IgG, IgA, IgM, IL-2, IL-4, IL-6 and specific antibody titer as well as the number of immunocompetent cells were systematically analyzed in the vaccinated mice. The results revealed that the levels of immunoglobulins, cytokines, the specific antibodies, together with the numbers of lymphocytes significantly increased in vaccinated mice inoculated with CNP-VPIL2 in contrast with those with naked IL-2 plasmids and blank plasmids. The CNP-VPIL2 immunized mice exhibited higher humoral and cellular immune responses, less severe clinical signs and lesions of disease caused by the bacteria than the other groups after challenge. These findings suggest that CNP-VPIL2 has a significant enhancement effect on immune responses of mice, which results in better immunoprotection against Salmonella infection, indicating that CNP-VPIL2 could be employed as an effective immunoadjuvant to elevate immunity of animals to conventional vaccines. PMID:17034859

Yang, Yi; Chen, Jianlin; Li, Hui; Wang, Yingyi; Xie, Zhao; Wu, Mei; Zhang, Huan; Zhao, Zhongzhong; Chen, Qian; Fu, Manliang; Wu, Kaiyuan; Chi, Cheng; Wang, Hongning; Gao, Rong



Levels of T-lymphocyte subpopulations, interleukin-1 beta, and soluble interleukin-2 receptor in acute myocardial infarction.  


T-lymphocyte levels may adversely affect the clinical course and outcome of patients with acute myocardial infarction (AMI). To characterize the T-lymphocyte profile during AMI and to explore whether these cells play a detrimental role in the extent of myocardial insult, levels of T-lymphocyte subpopulations, free soluble interleukin-2 receptor (sIL-2R), and interleukin-1 beta (IL-1 beta), were measured during the first week of AMI. Results were correlated with left ventricular ejection fraction (LVEF), age, sex, survival rate, thrombolytic therapy, and the occurrence of reinfarction. Thirty-nine patients, 20 men and 19 women aged 30 to 80 years, with first AMI were included. Patients were divided into two groups. Group A (13 patients) experienced reinfarction; group B (26 patients) did not. T-helper and-suppressor cells were measured by the indirect immunofluorescence method and sIL-2R and IL-1 serum levels by enzyme-linked immunosorbent assay (ELISA) methods on days 1, 4, and 7 after AMI. A low count of T-helper cells (CD4) was found on the first day after AMI in both AMI groups; however, the count returned to normal in group B but not in group A. A significant correlation (r = 0.63) was found between T-helper cell count on day 4 of AMI and LVEF assessed by radionuclide ventriculography, and between the CD4/CD8 ratio on day 1 and the creatine phosphokinase level (r = -0.6950). High sIL-2R levels were found in groups A and B of the AMI patients as compared with the control group (p < or = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8172050

Blum, A; Sclarovsky, S; Rehavia, E; Shohat, B



Requirements for interleukin 2 promoter transactivation by the Tax protein of human T-cell leukemia virus type 1.  

PubMed Central

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) upregulates the expression of several cellular genes by activating members of both the NF-kappaB and bZIP families of transcription factors. Recent studies demonstrate that the CD28 response element (CD28RE) of the interleukin 2 (IL-2) promoter is the site upregulated by Tax in stimulated T cells. Although some reports suggest that this site is transactivated by NF-kappaB family members, others disagree, leaving the identity of the transcription factor(s) binding the CD28RE unclear. The studies presented here further characterize the response of the IL-2 promoter and CD28RE to the HTLV-1 Tax protein and demonstrate that the TATA-proximal AP-1 binding site of the IL-2 promoter is also necessary for Tax transactivation in stimulated Jurkat cells. In contrast to its upregulation of the IL-2 promoter which requires T-cell stimulation, Tax transactivates the isolated CD28RE-AP-1 element without stimulation but is greatly synergized by calcium ionophore and phorbol ester. Additionally, transactivation of the IL-2 promoter requires the Tax activation domain involved in upregulation of bZIP-enhanced transcription while the NF-kappaB-activating domain of Tax is dispensable. Interestingly, both domains appear to be necessary for the activation of the isolated CD28RE-AP-1 sequence in the context of a heterologous promoter construct. This strongly suggests that activation of NF-kappaB is insufficient to activate transcription via the CD28RE-AP-1 element of the IL-2 promoter and that a different transcription factor, upregulated via the activation domain of the HTLV-1 Tax protein, may be involved.

Curtiss, V E; Smilde, R; McGuire, K L



Interleukin-2 signalling is modulated by a labile disulfide bond in the CD132 chain of its receptor  

PubMed Central

Certain disulfide bonds present in leucocyte membrane proteins are labile and can be reduced in inflammation. This can cause structural changes that result in downstream functional effects, for example, in integrin activation. Recent studies have shown that a wide range of membrane proteins have labile disulfide bonds including CD132, the common gamma chain of the receptors for several cytokines including interleukin-2 and interleukin-4 (IL-2 and IL-4). The Cys183–Cys232 disulfide bond in mouse CD132 is susceptible to reduction by enzymes such as thioredoxin (TRX), gamma interferon-inducible lysosomal thiolreductase and protein disulfide isomerase, which are commonly secreted during immune activation. The Cys183–Cys232 disulfide bond is also reduced in an in vivo lipopolysaccharide (LPS)-induced acute model of inflammation. Conditions that lead to the reduction of the Cys183–Cys232 disulfide bond in CD132 inhibit proliferation of an IL-2-dependent T cell clone and concomitant inhibition of the STAT-5 signalling pathway. The same reducing conditions had no effect on the proliferation of an IL-2-independent T cell clone, nor did they reduce disulfide bonds in IL-2 itself. We postulate that reduction of the Cys183–Cys232 disulfide in CD132 inhibits IL-2 binding to the receptor complex. Published data show that the Cys183–Cys232 disulfide bond is exposed at the surface of CD132 and in close contact with IL-2 and IL-4 in their respective receptor complexes. In addition, mutants in these Cys residues in human CD132 lead to immunodeficiency and loss of IL-2 binding. These results have wider implications for the regulation of cytokine receptors in general, as their activity can be modulated by a ‘redox regulator’ mechanism caused by the changes in the redox environment that occur during inflammation and activation of the immune system.

Metcalfe, Clive; Cresswell, Peter; Barclay, A. Neil



Carbon anhydrase IX specific immune responses in patients with metastatic renal cell carcinoma potentially cured by interleukin-2 based immunotherapy.  


The majority of clear-cell renal cell carcinomas (ccRCC) show high and homogeneous expression levels of the tumor associated antigen (TAA) carbonic anhydrase IX (CAIX), and treatment with interleukin-2 (IL-2) based immunotherapy can lead to cure in patients with metastatic renal cell carcinoma (mRCC). However, the involvement of CAIX specific CD8+ T cells and/or NK cells in the tumor eradication is unknown. We investigated T cell and antibody reactivity against overlapping 15-mer CAIX-peptides as well as HLA haplotype frequency and NK cell cytotoxicity in 11 patients with no evidence of disease (NED) following treatment with IL-2 based immunotherapy, and thus potentially cured. Immune reactivity in these patients was compared with samples from patients with dramatic tumor response obtained immediately at the cessation of therapy, samples from patients that experienced progressive disease during treatment and samples from healthy controls. We observed more focused but only weak and not consistent CAIX specific T-cells in the late observation and early observation response groups compared with the healthy control group. An increased frequency of the class II alleles HLA-DRB4 01:01, HLA-DPB 01:01 and HLA-DPB 03:01 was noted in the NED patients. In contrast, NK cytotoxicity was low even in the late observation response group as compared with controls. In particular, a HLA-B*40:01 restricted CD8+ T cell response recognizing the CAIX- derived peptide SEEEGSLKL was identified. This may have interest in future cancer vaccines, but more studies are needed to elucidate the immunological mechanisms of action in potentially cured patients treated with an immunotherapeutic agent. PMID:23802595

Rasmussen, Susanne; Donskov, Frede; Pedersen, Johannes W; Wandall, Hans H; Buus, Søren; Harndahl, Mikkel; Braendstrup, Peter; Claesson, Mogens H; Pedersen, Anders Elm



A monoclonal antibody recognizing an epitope shared by receptors for growth hormone, prolactin, interleukin 2 and interleukin 6.  


Monoclonal antibody (MAb) termed R7B4 was generated throughout the idiotypic-anti-idiotypic network from mice immunized with human and bovine growth hormones (GH). The Ab was selected on the basis that it did not recognize human GH (hGH) neither insolubilized nor in solution but inhibited 125I-hGH binding to receptors from rat and rabbit liver and from Nb2-cell membranes. Since it inhibited Nb2-cell mitogenesis stimulated by hGH, prolactins or placental lactogens, MAb R7B4 behaved as an antagonist of lactogenic hormones. Furthermore, the Ab impaired proliferative activity of interleukin 2 (IL-2) on Nb2 cells as well as growth of 7TD1 cells, an interleukin 6 (IL-6) dependent hybridoma not expressing GH receptors. Biotin-labeled MAb R7B4 specifically bound to rat liver microsomes, and the Ab was able to recognize Nb2 and 7TD1-cell membranes as shown by flow cytometry experiments. However, MAb binding was not hampered by hGH, indicating that the Ab did not mimic GH binding site to receptors. Immunoblot assays indicated that rat and rabbit liver as well as Nb2-cells membrane antigens recognized by MAb R7B4 were similar to those revealed by a MAb directed to prolactin receptors. In addition, MAb R7B4 was able to detect two bands probably corresponding to the somatogenic receptor in rabbit liver microsomes as well as three different proteins in 7TD1-cells showing molecular weights similar to those of the IL-6 receptor complex. Results suggest that MAb R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones, IL-2 and IL-6. To our knowledge, these data are the first experimental evidence of the existence of structural similarity between some of the receptors grouped in the cytokine receptor superfamily. PMID:10395088

Longhi, S A; Miranda, M E; Gobet, M G; Retegui, L A



Changes in thymic function in HIV-positive patients treated with highly active antiretroviral therapy and interleukin-2  

PubMed Central

Despite its potent antiviral activity, highly active antiretroviral therapy (HAART) only exerts a marginal effect on CD4+ T-cell regeneration in HIV-infected subjects. Combination therapies aimed at boosting T-cell activity and maturation may provide an important contribution to the restoration of immune function. Here, we report the results obtained by a two-year follow-up of a cohort of HIV-infected patients treated with a combination of HAART and interleukin-2 (IL-2). In these patients, in addition to a series of quantitative virological and immunological parameters, we investigated T-cell regeneration by an immunophenotypic assay monitoring CD4+ naïve T cells, and by analysis of thymic function, through the quantification of the excision DNA products of T-cell receptor rearrangement (TRECs) in lymphocytes. Compared with HAART alone, we found that the IL-2 combination therapy was equally effective in reducing the levels of viremia and marginally more effective in decreasing proviral DNA load. Strikingly, the IL-2 combination produced a marked increase in the number of CD4+ T cells bearing a naïve phenotype (CD45RA+, CD62L+), which was apparent for over 96 weeks after therapy. To assess whether these cells were the product of improved T-cell generation, we exploited a competitive quantitative molecular assay to quantify TRECs in peripheral blood lymphocytes. Surprisingly, we found that the levels of these molecules were unchanged in these patients. These findings indicate that improved thymic function does not account for the early rise of CD4 naïve cells in HIV-positive patients treated with IL-2, and suggest that alternative mechanisms of T-cell maturation and differentiation are responsible for this event.

De Paoli, P; Bortolin, M T; Zanussi, S; Monzoni, A; Pratesi, C; Giacca, M



Role of prolactin in the in vitro development of interleukin-2-driven anti-tumoural lymphokine-activated killer cells.  

PubMed Central

Exogenous prolactin (PRL) has been shown to synergize with low-dose interleukin-2 (IL-2) and induce the proliferation and lymphokine-activated killer (LAK) maturation of natural killer (NK) cells. PRL itself can also generate LAK activity. Here we show that its local production occurs during, and is necessary for, LAK development. IL-2-stimulated peripheral blood mononuclear cells (PBMC) and purified NK cells were exposed to anti-human (h)PRL antiserum, and residual LAK activity was measured on day 7 against the promyelocytic leukaemia cell line HL-60. Inhibition of LAK activity was much more evident in PBMC compared with NK cell cultures (47% decrease. P - 0.013 and 18.5% decrease. P = 0.048, respectively). Up-modulation of a 32S-methionine-labelled 27,000 MW protein was detected in the lysates and supernatants of IL-2-stimulated PBMC immunoprecipitated with an anti-PRL antiserum. By contrast, the cytoplasmic PRL immunoreactivity observed in freshly isolated NK cells and in IL-2-stimulated, but not unstimulated, NK cell cultures was not associated with PRL gene activation, and can thus be referred to internalized PRL. Preferential re-uptake of externally derived PRL by IL-2-stimulated NK cells was also indicated by up-modulation of the PRL receptor. These data, as a whole, indicate that the PRL promotion of LAK differentiation is mainly mediated by paracrine secretion, with a minor contribution from internalized PRL. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

Matera, L; Bellone, G; Lebren, J J; Kelly, P A; Hooghe Peters, E L; Di Celle, P F; Foa, R; Contarini, M; Avanzi, G; Asnaghi, V



IL2RA Genetic Heterogeneity in Multiple Sclerosis and Type 1 Diabetes Susceptibility and Soluble Interleukin-2 Receptor Production  

PubMed Central

Multiple sclerosis (MS) and type 1 diabetes (T1D) are organ-specific autoimmune disorders with significant heritability, part of which is conferred by shared alleles. For decades, the Human Leukocyte Antigen (HLA) complex was the only known susceptibility locus for both T1D and MS, but loci outside the HLA complex harboring risk alleles have been discovered and fully replicated. A genome-wide association scan for MS risk genes and candidate gene association studies have previously described the IL2RA gene region as a shared autoimmune locus. In order to investigate whether autoimmunity risk at IL2RA was due to distinct or shared alleles, we performed a genetic association study of three IL2RA variants in a DNA collection of up to 9,407 healthy controls, 2,420 MS, and 6,425 T1D subjects as well as 1,303 MS parent/child trios. Here, we report “allelic heterogeneity” at the IL2RA region between MS and T1D. We observe an allele associated with susceptibility to one disease and risk to the other, an allele that confers susceptibility to both diseases, and an allele that may only confer susceptibility to T1D. In addition, we tested the levels of soluble interleukin-2 receptor (sIL-2RA) in the serum from up to 69 healthy control subjects, 285 MS, and 1,317 T1D subjects. We demonstrate that multiple variants independently correlate with sIL-2RA levels.

Cooper, Jason; Downes, Kate; Anderson, David E.; Severson, Christopher; Clark, Pamela M.; Healy, Brian; Walker, Neil; Aubin, Cristin; Oksenberg, Jorge R.; Hauser, Stephen L.; Compston, Alistair; Sawcer, Stephen; De Jager, Philip L.; Wicker, Linda S.



Inhibition of the calcitonin-induced outward current in identified Aplysia neurons by interleukin-1 and interleukin-2.  


1. The effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the calcitonin (CT)-induced outward current recorded from identified neurons (R9-R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. 2. Micropressure ejection of CT onto the soma of the neuron induced a slow outward current [Io(CT); 4-6 nA in amplitude, 30-40 sec in duration] associated with a decrease in input membrane conductance. 3. Io(CT) was increased by hyperpolarization. 4. The extrapolated reversal potential was +10 mV. Additionally, Io(CT) was sensitive to changes in (Na+)o but not to changes in (K+)o, (Ca2+)o, and (Cl-)o. 5. Micropressure-ejected forskolin produced a slow outward current similar to that induced by CT. 6. Bath-applied rhIL-1 and rhIL-2 (10-40 U/ml) reduced the CT-induced current in identified Aplysia neurons without affecting the resting membrane conductance or the holding current. 7. The inhibitory effects of both cytokines on the current were completely reversible. Heat-inactivated rhIL-1 and rhIL-2 were without effect. 8. These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the CT-induced outward current associated with a decrease in Na+ conductance in the nervous system of Aplysia. Therefore, the study suggests that these cytokines may also serve as neuromodulators. PMID:7842475

Sawada, M; Ichinose, M; Stefano, G B



The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma  

Microsoft Academic Search

The recent use of interleukin 2 (IL-2) and interleukin 4 (IL-4) single cytokine modified tumour cells in rodent models has demonstrated a potential use of these cytokines to produce autologous cancer cell vaccines. Here we compare the potential therapeutic benefit of transduction with IL-2 or IL-4 alone, and combined IL-2 + IL-4 in B16F10 cells, a murine malignant melanoma of

SJ Hollingsworth; D Darling; J Gäken; W Hirst; P Patel; M Kuiper; P Towner; S Humphreys; F Farzaneh; GJ Mufti



Locoregional therapy with polyethylene-glycol-modified interleukin-2 of an intradermally growing hepatocellular carcinoma in the guinea pig induces T-cell-mediated antitumor activity  

Microsoft Academic Search

Therapy with repeated intratumoral and perilymphatic administration of relatively low doses of polyethylene-glycol(PEG)-modified interleukin-2 (IL-2) in the syngeneic guinea pig line 10 (L10) hepatocarcinoma results in significant local tumor growth inhibition and a delay in development of regional lymph node metastases of more than 3 weeks when compared to controls. Occasionally animals are cured of tumor. The mechanism of this

L. T. M. Balemansl; V. Mattijssen; P. A. Steerenberg; B. E. M. Van Driel; P. H. M. De Mulder; W. Den Otter



Successful in vivo blockade of CD25 (high-affinity interleukin 2 receptor) on T cells by administration of humanized anti-Tac antibody to patients with psoriasis  

Microsoft Academic Search

Background: Daclizumab is a humanized antibody to the ?-subunit (CD25) of the interleukin 2 (IL-2) receptor that blocks normal IL-2 binding to this receptor. Because IL-2 is a major stimulus for T-cell growth, blockade of the IL-2 receptor could be useful in treating T-cell-mediated (autoimmune) diseases. Objective: Our purpose was to determine whether adequate concentrations of antibody were achieved in

James G. Krueger; Ian B. Walters; Megumi Miyazawa; Patricia Gilleaudeau; John Hakimi; Susan Light; Amelia Sherr; Alice B. Gottlieb



Anti-Tac-H, a Humanized Antibody to the Interleukin 2 Receptor with New Features for Immunotherapy in Malignant and Immune Disorders  

Microsoft Academic Search

The M, 55,000 interleukin 2 receptor peptide (Tac; CD25) is not expressed by normal resting T-cells but is markedly up-regulated in adult T-cell leukemia and other malignancies, as well as on T-cells activated in normal immune, autoimmune, allograft, and graft-versus-host settings. Anti-Tac is a mouse monoclonal antibody directed against the Tac peptide. Our prior attempts to use this antibody in

R. P. Junghans; T. A. Waldmann; N. F. Landolfi; N. M. Avdalovic; W. P. Schneider; C. Queen



Structure--Function Relationships for the Interleukin 2 Receptor: Location of Ligand and Antibody Binding Sites on the Tac Receptor Chain by Mutational Analysis  

Microsoft Academic Search

The Tac protein plays a role in high- and low-affinity interleukin 2 (IL-2) receptors. A mutational survey of this molecule identified several small segments in which the binding of IL-2 was particularly sensitive to amino acid substitutions. Two of the segments (residues 1-6 and 35-43) located in the exon 2-encoded region of the molecule overlapped the apparent binding sites of

Richard J. Robb; Cynthia M. Rusk; Michael P. Neeper



Characterisation and expression analysis of interleukin 2 (IL2) and IL21 homologues in the Japanese pufferfish, Fugu rubripes , following their discovery by synteny  

Microsoft Academic Search

This investigation provides the first conclusive evidence for the existence of the interleukin 2 (IL-2) and IL-21 genes in bony fish. The IL-2 and IL-21 sequences have been determined in Fugu rubripes by exploiting the conservation of synteny that is found between regions of the human and Fugu genomes. The predicted 149-amino acid IL-2 homologue contains the IL-2 family signature,

Steve Bird; Jun Zou; Tomoya Kono; Masahiro Sakai; Johannes Martinus Dijkstra; Chris Secombes



Beta-interferon and interleukin-2 prolong more than three times the survival of 26 consecutive endocrine dependent breast cancer patients with distant metastases: an exploratory trial  

Microsoft Academic Search

Distant metastases from breast cancer are incurable. In endocrine-responsive patients antiestrogens are commonly administered as first and second line therapy. Regrettably, tumor growth becomes resistant to this relatively innocuous therapy. Beta-interferon was unsuccessfully added to tamoxifen to induce estrogen receptor enhancement. In mice, interleukin-2 added to tamoxifen increased their mutual anti-tumor activities. Nevertheless, no effective clinical application has been developed.

Andrea Nicolini; Angelo Carpi



The release of interleukin-2 (IL2) and colony stimulating activity (CSA) in aplastic anemia patients: opposite behaviour with improvement of bone marrow function  

Microsoft Academic Search

Peripheral blood cells from patients with aplastic anemia were tested for their ability to release interleukin-2 (IL-2) and colony stimulating activity (CSA) before treatment. IL-2 release — as measured in the mouse thymocyte assay — was abnormally high in 18\\/34, and abnormally low in 10\\/34 patients. “Low” release was due to simultaneous release of thymocyte inhibitors. In 18 patients who

Catherine Nissen; Yolanda Moser; Johanna Weis; Andreas Wtirsch; Alois Gratwohl; Bruno Speck



The Src-Family Kinase, Fyn, Regulates the Activation of Phosphatidyllnositol 3Kinase in an Interleukin 2-responsive T Cell Line  

Microsoft Academic Search

$ummaary The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein ryrosine kinase (FFK) activity. One IL-2-regulated FTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol

Larry M. Karnitz; Shaft L. Sutor; Robert T. Abraham


The inhibitory effect of common traditional anti-rheumatic herb formulas on prostaglandin E and interleukin 2 in vitro: a comparative study with Tripterygium wilfordii  

Microsoft Academic Search

To understand the clinical efficacy of traditional anti-rheumatic herbal medicines on acute and severe arthritis or immune diseases, four herbal formulas and one herb were tested in vitro to determine their effects on prostaglandin E2 (PGE2) and interleukin 2 (IL2). Peripheral blood mononuclear cells from healthy subjects were incubated with different concentrations of four herbal formulas including Shaur Yau Gan

C. T Chou; S. C Chang



Elimination of Established Liver Métastases by Human Interleukin 2-activated Natural Killer Cells after Locoregional or Systemic Adoptive Transfer1  

Microsoft Academic Search

An in vivo model of liver metastasis induced by human gastric carci noma was established in nude mice and used for locoregional or systemic immunotherapy with a subset of human A-natural killer (NK) cells de fined previously. A single intrasplenic (i.s.) delivery of A-NK cells (1 x llf i and interleukin 2 (IL-2; 60,000 international units, twice a day for

Kazuhiko Okada; Ulf Nannmark; Nikola L. Vujanovic; Simon Watkins; Per Basse; Ronald B. Herberman; Theresa L. Whiteside


Effective Genetic Therapy of Established Medullary Thyroid Carcinomas with Murine Interleukin2: Dissemination and Cytotoxicity Studies in a Rat Tumor Model  

Microsoft Academic Search

Replication-defective adenovirus (AdCMVmIL2) expressing mu- rine interleukin-2 was directly injected into rat medullary thyroid carcinomas to examine antitumor activity. AdCMVmIL2 cured 42.9% of all treated tumor bearing animals. Most cured rats were protected against tumor growth after subsequent rechallenge with wild-type tumor cells, reflecting the immunity obtained from the original treat- ment. Studies of viral dissemination showed that the intratumoral




The Immediate-Early Gene Product Egr1 Regulates the Human Interleukin2 Receptor b-Chain Promoter through Noncanonical Egr and Sp1 Binding Sites  

Microsoft Academic Search

The interleukin-2 IL-2 receptor b-chain (IL-2Rb) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rb is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides 2170 and 2139 of the human




Monocytes and neutrophils as ‘bad guys’ for the outcome of interleukin-2 with and without histamine in metastatic renal cell carcinoma – results from a randomised phase II trial  

Microsoft Academic Search

Histamine (HDC) inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer (NK) and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). We have explored this potential mechanism clinically in two randomised phase II trials in metastatic renal cell carcinoma (mRCC). In parallel with the clinical

F Donskov; M Hokland; N Marcussen; H H Torp Madsen; H von der Maase



Association of interleukin-2 and interferon-? production by blood mononuclear cells in infancy with parental allergy skin tests and with subsequent development of atopy  

Microsoft Academic Search

The mechanisms regulating the onset of atopic sensitization in human beings are not yet fully clarified. We assessed the capacity of mitogen-stimulated umbilical and peripheral blood mononuclear cells to produce interferon-? (IFN-?) and interleukin-2 (IL-2) at birth and at 9 months of age in 159 infants. Mononuclear cell production of both IFN-? and IL-2 at 9 months, but not at

Fernando D. Martinez; Debra A. Stern; Anne L. Wright; Catharine J. Holberg; Lynn M. Taussig; Marilyn Halonen



Treatment of recurrent malignant glioma by repeated intracerebral injections of human recombinant interleukin-2 alone or in combination with systemic interferon-?. Results of a phase I clinical trial  

Microsoft Academic Search

Nine patients with a recurrent malignant glioma were treated with repeated intracavitary or intracerebroventricular injections of human recombinant interleukin-2 (rIL-2) alone or in combination with systemic interferon-a (IFN-a). Five patients received only rIL-2 and four were treated with rIL-2 plus subcutaneous injections of IFN-a. Therapy was administered on a Monday, Wednesday, Friday schedule for up to 10 weeks, beginning with

Randall E. Merchant; Daniel W. McVicar; Lynn H. Merchant; Harold F. Young



Production and characterization of a bicistronic Moloney-based retroviral vector expressing human interleukin 2 and herpes simplex virus thymidine kinase for gene therapy of cancer  

Microsoft Academic Search

Gene-based therapeutic strategies for cancer mainly include augmentation of immunotherapeutic and chemotherapeutic approaches. In this study we report the design and functional assay of a novel bicistronic Moloney-based retroviral vector expressing human interleukin-2 (IL-2) and herpesvirus thymidine kinase (tk) through a cap-dependent translation and an internal ribosome entry site (IRES)-regulated translation, respectively. This construct has the potential for allowing combination

M Pizzato; E Franchin; P Calvi; R Boschetto; M Colombo; S Ferrini; G Palù



Infection by Mink Cell Focus-Forming Viruses Confers Interleukin 2 (IL2) Independence to an IL2Dependent Rat T-Cell Lymphoma Line  

Microsoft Academic Search

The development of T-cell lymphomas in rodents infected with type C retroviruses has been linked to the generation of a class of envelope (env) recombinant viruses called mink cell focus-forming viruses (MCF viruses) in the preleukemic thymus. To determine whether infection by MCF viruses altered the growth phenotype of retrovirus-induced T-cell lymphomas, a Moloney murine leukemia virus-induced interleukin-2 (IL-2)-dependent rat

Philip N. Tsichlis; Susan E. Bear



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


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 inhibition in murine models are now being tested by the Cytokine Working Group in patients with advanced renal cell carcinoma. PMID:22660171

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



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

SciTech Connect

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

Puri, R.K.; Travis, W.D.; Rosenberg, S.A. (CBER, FDA, Bethesda, MD (USA))



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

SciTech Connect

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.

Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H. (Tokyo Women's Medical College (Japan))



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

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.

Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P. (Institut National Pour la Recherche Scientifique et Medicale U.211 (France))



Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition  

PubMed Central

We developed a method for production of antigen-specific, H-2-restricted T cell hybrids. The tumor cell partner in the fusions was itself a T cell hybrid, FS6-14.13.AG2 (or its derivatives), which could be induced to produce the growth factor, interleukin-2 (IL-2), in response to a challenge with concanavalin A, but had no known antigen specificity. The normal T cell partner in the fusions was a population of lymph node T cell blasts that had been highly enriched in antigen-specific, H-2-restricted T cells by in vivo immunization, followed by in vitro challenge with antigen and clonal expansion in IL-2-containing medium. These fusions produced hybrids that grew constitutively in culture. A sizable proportion of the hybrids demonstrated the ability to produce IL-2 in response to a challenge with specific antigen presented by irradiated spleen cells of the appropriate H-2 type. Four cloned antigen/H-2-specific hybrid lines were produced. AO-40.10 responded to chicken ovalbumin (OVA) when presented by I-A(k)-bearing cells. DC1.18.3 responded to the apo form of beef cytochrome c when presented with I-A(d). AODK-10.4 responded to keyhole limpet hemocyanin (KLH) presented with I-A (d). AODK-1.16 also responded to KLH presented by a product of the I region of H-2(d), but the data were consistent with either a product of the I-J-I-E(d) region or a combinatorial molecule with elements from both I-A(d) and I-E(d)/I-C(d). Coincidentally, AO-40.10 was shown to have an unexpected alloreactivity with a product of H-2(b) mapping to the K-I-A region. These hybrids should prove invaluable as sources of monoclonal material for the study of the receptor(s) on T cells with H-2-restricted antigen specificities. We also generated T cell hybrids with two antigen/H-2 specificities by fusing an azaguanine-resistant clone of AO-40.10 to normal T cells with a different antigen/H-2 specificity. Many of the hybrids retained reactivity to OVA plus H-2(a) and to the second antigen/H-2 combination. None reacted to either OVA plus the second H-2 type or to the second antigen plus H-2(a). One of these hybrids was successfully cloned to produce the line AOFK- 11.11.1. It retained the ability to recognize OVA plus I-A(k) inherited from one parent, and KLH plus IA(f) inherited from the other. It did not recognize OVA plus IA(f) or KLH plus I-A(k). These results have some bearing on models describing the nature of T cell receptors for antigen recognized in association with H-2 products. They do not support models in which antigen and H-2 are recognized separately by two independent T cell receptors.

Kappler, JW; Skidmore, B; White, J; Marrack, P



A direct comparison of immunological and clinical effects of interleukin 2 with and without interferon-alpha in humans.  


Interleukin 2 (IL-2) and interferon-alpha (IFN-alpha) are cytokines with synergistic antitumor effects in mouse models. The biological effects of this combination, however, have not been directly compared to each agent alone in humans. We conducted a Phase 1B trial of IL-2 plus or minus IFN-alpha in 38 cancer patients. The objectives of this trial were to determine which doses of IFN-alpha and IL-2 maximally enhanced biological responses, and to determine whether the combined administration of IFN-alpha and IL-2 would result in a potentiation of biological responses over IL-2 alone. Patients received 4 days of IL-2 (1.5 x 10(6) units/m2/day or 3.0 x 10(6) units/m2/day) as a continuous infusion followed by a 3-day rest period, weekly for 3 weeks, with a 3-week rest period between 2 treatment courses. IFN-alpha (0.5 x 10(6) or 5 x 10(6) units/m2/day) was administered s.c. on days 1-4 weekly for 3 weeks with one of the 3-week courses. Patients were randomized to receive either IL-2 alone for course 1, followed by IL-2/IFN-alpha for course 2, or IL-2/IFN-alpha in course 1, followed by IL-2 alone. Immunological parameters were evaluated before treatment, and 24 h after completion of the third week of IL-2. A statistically significant increase in the percentage of circulating natural killer cells (CD56), natural killer cells bearing the Fc receptor (CD16), and activated T cells (CD25) was observed following IL-2 alone, and following IL-2 plus IFN-alpha. Significant increases in lymphocyte-activated killer cell cytotoxicity, antibody cellular cytotoxicity, and serum IL-2 receptor were also observed following both IL-2 and IL-2 plus IFN-alpha. However, no significant differences were observed in the magnitude of the increase in the IL-2-alone group when compared to the IL-2 plus IFN-alpha group. The mean fluorescent intensity of monocytes positive for HLA-DR and Fc receptor expression also increased significantly in both groups, as did serum beta 2-microglobulin expression and indoleamine 2,3-dioxygenase activity. However, increases were not significantly different between patients receiving IL-2 alone and IL-2 plus IFN-alpha. No dose response effect for IFN-alpha was observed for any of the parameters assessed. Toxicities consisted primarily of constitutional toxicities, including fever, rigors, malaise, headache, anorexia, and a decrease in performance status. No clinically significant differences in toxicities were observed between courses consisting of IL-2 and those consisting of IFN-alpha and IL-2.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8443808

Schiller, J H; Hank, J; Storer, B; Borchert, A A; Moore, K H; Albertini, M; Bechhofer, R; Wesley, O; Brown, R R; Bastin, A M



Cytokine mRNA levels in human fat tissue after injection lipolysis with phosphatidylcholine and deoxycholate.  


Injections with Lipostabil, a phosphatidylcholine and deoxycholate containing substance, have become a popular technique to treat localized fat accumulation and lipomas. It is believed that the injected substance leads to fat cell destruction with subsequent acute panniculitis followed by a repair process of the treated fat tissue. We sought to investigate the mRNA expression of cytokines within the acute stage of panniculitis following Lipostabil injections. Tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-8, and IL-10 mRNA expression were determined using quantitative real-time reverse transcriptase polymerase chain reaction in treated and adjacent non-treated fat tissue of lipomas 48 h after injection in seven patients. Following injection lipolysis with Lipostabil, TNF-alpha, IL-6, IL-8, and IL-10 mRNA levels were significantly elevated in treated fat tissue compared to non-treated fat. No significant differences were found for IL-4. Both in treated and non-treated fat tissue, mRNA of IFN-gamma, IL-2, and IL-5 was not expressed. Injection lipolysis induces acute panniculitis within the treated fat tissue, associated with changes of the cytokine profile. However, their pathogenetic relevance with respect to clinical dissolving of fat needs further investigation. PMID:18563422

Bechara, Falk G; Skrygan, Marina; Kreuter, Alexander; Altmeyer, Peter; Gambichler, Thilo



Sputum Mycobacterium tuberculosis mRNA as a marker of bacteriologic clearance in response to antituberculosis therapy.  


mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability. PMID:19923475

Li, L; Mahan, C S; Palaci, M; Horter, L; Loeffelholz, L; Johnson, J L; Dietze, R; Debanne, S M; Joloba, M L; Okwera, A; Boom, W H; Eisenach, K D



rpt-1, an intracellular protein from helper/inducer T cells that regulates gene expression of interleukin 2 receptor and human immunodeficiency virus type 1.  

PubMed Central

The Rpt-1 (for regulatory protein, T-lymphocyte, 1) gene, selectively expressed by resting but not by activated CD4+ inducer T cells, encodes an intracellular protein (rpt-1, Mr 41,000) that down-regulates gene expression directed by the promoter region of the gene encoding interleukin 2 receptor alpha chain and by the long terminal repeat of human immunodeficiency virus type 1. The data reported here suggest that rpt-1 levels may be inversely correlated with activation of CD4+ T cells and human immunodeficiency virus replication leading to clinical symptoms of the acquired immunodeficiency syndrome. Images

Patarca, R; Freeman, G J; Schwartz, J; Singh, R P; Kong, Q T; Murphy, E; Anderson, Y; Sheng, F Y; Singh, P; Johnson, K A



Evaluation of murine lymphocyte membrane potential, intracellular free calcium, and interleukin-2 receptor expression upon exposure to 1,1-dimethylhydrazine.  


The effects of 1,1-dimethylhydrazine (UDMH) on several early events associated with lymphocyte activation were examined. The concentration of intracellular calcium ([Ca2+]i) and membrane potential of murine lymphocytes were found to be altered upon exposure to UDMH; [Ca2+]i was increased in murine thymocytes, while splenocytes exhibited membrane hyperpolarization. In addition, interleukin-2 receptor expression induced by in-vitro concanavalin A stimulation of murine splenocytes at 24 and 48 h in the presence of UDMH was not affected. UDMH may interfere with the ability of these two distinct lymphocyte populations to regulate normal ionic fluctuations, thus contributing to altered immune responsiveness. PMID:1609436

Frazier, D E; Tarr, M J; Olsen, R G



[A new crystal form of the Fab fragment of a monoclonal antibody to human interleukin-2: the three-dimensional structure at 2.7 A resolution].  


The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 in a new crystal form (space group P2(1)2(1)2(1); unit cell parameters: a = 42.82, b = 90.68, and c = 139.82 A) was determined by the X-ray molecular replacement method at the resolution of 2.7 A. The protein folding and the stereochemistry of its antigen-binding site were comparatively analyzed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // PMID:15562966

Pletnev, V Z; Goriacheva, E A; Tsygannik, I N; Nesmeianov, V A; Pletnev, S V; Pangborn, W; Daux, W


Estrogen upregulates cyclic AMP response element modulator ? expression and downregulates interleukin-2 production by human T lymphocytes.  


Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex multifactorial pathogenesis. T lymphocytes play a critical role in disease pathogenesis and display abnormal gene expression and poor interleukin (IL)-2 production. We previously showed that the expression of the transcriptional repressor cyclic AMP response element modulator ? (CREM?) is increased in SLE T cells and contributes to reduced IL-2 production. Although estrogen is implicated in the onset and exacerbation of SLE, the precise nature of molecular events regulated by estrogen in immune cell function is not well understood. Here, we asked whether estrogen regulates the expression of CREM? in human T lymphocytes. We show that exposure of human T cells to 17-?-estradiol leads to a dose-dependent increase in CREM? mRNA expression, and this increase appears to be mediated through the estrogen receptors ? and ?. We show that the increased expression of CREM? is due to increased transcriptional activity of the CREM promoter and is mediated by increased expression and binding of the Sp1 transcriptional activator. We further show that estrogen treatment leads to a dose-dependent decrease in IL-2 mRNA and cytokine production by T cells. Finally, the effect of ?-estradiol on CREM? is observed more frequently in T cells from women than from men. We conclude that estrogen can modulate the expression of CREM? and lead to IL-2 suppression in human T lymphocytes, thus revealing a molecular link between hormones and the immune system in SLE. PMID:22281835

Moulton, Vaishali R; Holcomb, Dana R; Zajdel, Melissa C; Tsokos, George C



Severe Adverse Events from the Treatment of Advanced Melanoma: A Systematic Review of Severe Side Effects Associated with Ipilimumab, Vemurafenib, Interferon Alfa-2b, Dacarbazine, and Interleukin-2.  


Abstract Background: Current immunomodulatory agents for stage III and IV melanoma exert different mechanisms of action that manifest in distinct adverse events. Objective: This systematic review aims to synthesize safety data from clinical trials on ipilimumab, vemurafenib, interferon alfa-2b, dacarbazine, and interleukin-2 to elucidate the severe adverse events associated with each melanoma therapy. Methods: Through a systematic search using MEDLINE, EMBASE, and the Cochrane Central Register between January 1, 2010 and June 1, 2012, we identified 32 clinical trials with 5802 subjects that met inclusion criteria. Results: Ipilimumab was associated with immune-mediated diarrhea and colitis, with an incidence rate of 0.0017 cases per 100 person-years. Patients receiving vemurafenib developed keratoacanthomas and cutaneous squamous cell carcinoma at an incidence rate of 0.0025 cases per 100 person-years. Treatment with interferon alfa-2b precipitated depression at an incidence rate of 0.0002 cases per 100 person-years. Dacarbazine was associated with respiratory toxicity and dyspnea, with incidence rates of 0.0001 and 0.00008 cases per 100 person-years, respectively. Interleukin-2 treatment induced vascular leak syndrome, with symptoms of hypotension and oliguria observed at incidence rates of 0.17 and 0.15 cases per 100 person-years, respectively. Findings may serve as a foundation for further research and guide clinical recommendations. PMID:23763243

Ma, Chelsea; Armstrong, April



An Efficient Method for the Delivery of the Interleukin-2 Gene to Human Hematopoietic Cells using the ?Fiber-Modified Recombinant Adenovirus  

PubMed Central

Recombinant human adenovirus serotype 5 (Ad5/35F-IL2) with modified fibres containing the C-terminal domain fiber-knob of human adenovirus serotype 35, carrying the gene of recombinant human IL-2, has been designed. As a result of the fiber modification, the adenovirus can efficiently deliver the genetic information to bone marrow leukocytes and the tumor blood cells KG-1A (human myeloblastic leukemia cells) and U937 (human histiocytic lymphoma cells), which are normally resistant to Ad5 infection. The flow cytometry data reveal that the modified Ad5/35F penetrates into a population of monocytes, granulocytes, and blast cells of human bone marrow. The expression of interleukin-2 in CAR-negative bone marrow leukocytes (3682.52 ± 134.21 pg/ml) and the cell lines KG-1A (748.3 ± 32.8 pg/ml) and U937 (421.5 ± 59.4 pg/ml) transduced with adenovirus Ad5/35F-IL2 is demonstrated. The fiber-modified adenovirus can be used as a vector for the efficient gene delivery of interleukin-2 to human normal and tumor hematopoietic cells.

Rogozhin, V.N.; Logunov, D.Yu.; Shchebliakov, D.V.; Shmarov, M.M.; Khodunova, E.E.; Galtseva, ?I.V.; Belousova, R.V.; Naroditsky, B.S.; Gintsburg, A.L.



Human intestinal epithelial cells express functional cytokine receptors sharing the common gamma c chain of the interleukin 2 receptor.  

PubMed Central

Interleukin (IL) 2 signaling requires the dimerization of the IL-2 receptor beta (IL-2R beta) and common gamma (gamma c) chains. The gamma is also a component of the receptors for IL-4, IL-7, and IL-9. To assess the extent and role of the receptor signal transducing system utilizing the gamma c chain on human intestinal epithelial cells, the expression of gamma c, IL-2R beta, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by reverse transcription-coupled PCR on human intestinal epithelial cell lines and on isolated primary human intestinal epithelial cells. Caco-2, HT-29, and T-84 cells were found to express transcripts for the gamma c and IL-4R chains constitutively. IL-2R beta chain expression was demonstrated in Caco-2 and HT-29 but not in T-84 cells. None of the cell lines expressed mRNA for the IL-2R alpha chain. After stimulation with epidermal growth factor for 24 h Caco-2, HT-29, and T-84 cells expressed transcripts for IL-7R. In addition, Caco-2 and HT-29 cells expressed mRNA for the IL-9R. Receptors for IL-2, IL-4, IL-7, and IL-9 on intestinal epithelial cells lines appeared to be functional; stimulation with these cytokines caused rapid tyrosine phosphorylation of proteins. The relevance of the observations in intestinal epithelial cell lines for intestinal epithelial function in vivo was supported by the demonstration of transcripts for gamma c, IL-2R beta, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

Reinecker, H C; Podolsky, D K



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


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

Schwinzer, R; Siefken, R



Analysis of activated T cell infiltrates in rat renal allografts by gamma camera imaging after injection of 123iodine-interleukin 2.  


Activated T cells bearing receptors for interleukin 2 (IL-2) play an important role in immunity and in immunopathological processes such as allograft rejection. In order to investigate the presence of activated T cells in lymphocytic infiltrates in transplanted kidneys, we investigated the uptake and retention of radioactivity after an intravenous injection of radioiodinated IL-2 in experimentally transplanted rats. IL-2 was enzyme radiolabelled with 123iodine using a glucose oxidase/lactoperoxidase method and shown to retain specific binding on an IL-2 receptor positive cell line, C58E6. To examine the kinetics of 123iodine-interleukin 2 (123I-IL-2) uptake in vivo, animals that had been transplanted five days previously with allogeneic or syngeneic grafts were injected with 123I-IL-2 and then imaged using an external gamma camera. Radioactivity was measured at time points up to 240 min after intravenous injection of 123I-IL-2. Four groups of animals were examined: allogeneic grafts (n = 7); syngeneic grafts (n = 6); ischaemic native kidneys (n = 5) all following injection with 123I-IL-2; and allogeneic transplants (n = 5) after injection of 123I-lactalbumin, an irrelevant molecule of similar molecular weight to IL-2. The peak radioactivity after injection was measured and the amount of radioactivity retained in the graft at increasing time intervals after injection was expressed as a function of initial peak radioactivity. At four hours after injection of 123I-IL-2, mean retention of activity by rejecting grafts was 77(+/- 2.68)% of peak activity, compared to 45(+/- 6.38)% in syngeneic controls (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8081762

Abbs, I C; Pratt, J R; Dallman, M J; Sacks, S H



Molecular Characteristics of CTA056, a Novel Interleukin-2-Inducible T-Cell Kinase Inhibitor that Selectively Targets Malignant T Cells and Modulates Oncomirs  

PubMed Central

Interleukin-2-inducible T-cell kinase (Itk) is a member of the Btk (Bruton's tyrosine kinase) family of tyrosine kinases. Itk plays an important role in normal T-cell functions and in the pathophysiology of both autoimmune diseases and T-cell malignancies. Here, we describe the initial characterization of a selective inhibitor, 7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (CTA056), that was developed through screening a 9600-compound combinatorial solution phase library, followed by molecular modeling, and extensive structure-activity relationship studies. CTA056 exhibits the highest inhibitory effects toward Itk, followed by Btk and endothelial and epithelial tyrosine kinase. Among the 41 cancer cell lines analyzed, CTA056 selectively targets acute lymphoblastic T-cell leukemia and cutaneous T-cell lymphoma. Normal T cells are minimally affected. Incubation of Jurkat and MOLT-4 cells with CTA056 resulted in the inhibition of the phosphorylation of Itk and its effectors including PLC-?, Akt, and extracellular signal-regulated kinase, as well as the decreased secretion of targeted genes such as interleukin-2 and interferon-?. Jurkat cells also underwent apoptosis in a dose-dependent manner when incubated with CTA056. The potent apoptosis-inducing potential of CTA056 is reflected by the significant modulation of microRNAs involved in survival pathways and oncogenesis. The in vitro cytotoxic effect on malignant T cells is further validated in a xenograft model. The selective expression and activation of Itk in malignant T cells, as well as the specificity of CTA056 for Itk, make this molecule a potential therapeutic agent for the treatment of T-cell leukemia and lymphoma.

Guo, Wenchang; Liu, Ruiwu; Ono, Yoko; Ma, Ai-Hong; Martinez, Anthony; Sanchez, Eduardo; Wang, Yan; Huang, Wenzhe; Mazloom, Anisha; Li, Jixian; Ning, Jinying; Maverakis, Emanual; Lam, Kit S.



Dacarbazine and interferon ? with or without interleukin 2 in metastatic melanoma: a randomized phase III multicentre trial of the Dermatologic Cooperative Oncology Group (DeCOG)  

PubMed Central

In several phase II-trials encouraging tumour responses rates in advanced metastatic melanoma (stage IV; AJCC-classification) have been reported for the application of biochemotherapy containing interleukin 2. This study was designed to compare the efficacy of therapy with dacarbazine (DTIC) and interferon ? (IFN-?) only to that of therapy with DTIC and IFN-? with the addition of interleukin 2 (IL-2) in terms of the overall survival time and rate of objective remissions and to provide an elaborated toxicity profile for both types of therapy. 290 patients were randomized to receive either DTIC (850?mg/m2every 28 days) plus IFN-?2a/b (3?MIU/m2, twice on day 1, once daily from days 2 to 5; 5 MIU/m23 times a week from week 2 to 4) with or without IL-2 (4.5?MIU/m2for 3 hours i.v. on day 3; 9.0?MIU/m2i.v. day 3/4; 4.5?MIU/m2s.c. days 4 to 7). The treatment plan required at least 2 treatment cycles (8 weeks of therapy) for every patient. Of 290 randomized patients 281 were eligible for an intention-to-treat analysis. There was no difference in terms of survival time from treatment onset between the two arms (median 11.0 months each). In 273 patients treated according to protocol tumour response was assessable. The response rates did not differ between both arms (P = 0.87) with 18.0% objective responses (9.7% PR; 8.3% CR) for DTIC plus IFN-? as compared to 16.1% (8.8% PR; 7.3% CR) for DTIC, IFN-? and IL-2. Treatment cessation due to adverse reactions was significantly more common in patients receiving IL-2 (13.9%) than in patients receiving DTIC/IFN-? only (5.6%). In conclusion, there was neither a difference in survival time nor in tumour response rates when IL-2, applied according to the combined intravenous and subcutaneous schedule used for this study, was added to DTIC and IFN-?. However, toxicity was increased in melanoma patients treated with IL-2. Further phase III trials with continuous infusion and higher dosages must be performed before any final conclusions can be drawn on the potential usefulness of IL-2 in biochemotherapy of advanced melanoma. © 2001 Cancer Research Campaign

Hauschild, A; Garbe, C; Stolz, W; Ellwanger, U; Seiter, S; Dummer, R; Ugurel, S; Sebastian, G; Nashan, D; Linse, R; Achtelik, W; Mohr, P; Kaufmann, R; Fey, M; Ulrich, J; Tilgen, W



Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene.  


The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor. PMID:8943338

Lécine, P; Algarté, M; Rameil, P; Beadling, C; Bucher, P; Nabholz, M; Imbert, J



Dacarbazine and interferon alpha with or without interleukin 2 in metastatic melanoma: a randomized phase III multicentre trial of the Dermatologic Cooperative Oncology Group (DeCOG).  


In several phase II-trials encouraging tumour responses rates in advanced metastatic melanoma (stage IV; AJCC-classification) have been reported for the application of biochemotherapy containing interleukin 2. This study was designed to compare the efficacy of therapy with dacarbazine (DTIC) and interferon alpha (IFN-alpha) only to that of therapy with DTIC and IFN-alpha with the addition of interleukin 2 (IL-2) in terms of the overall survival time and rate of objective remissions and to provide an elaborated toxicity profile for both types of therapy. 290 patients were randomized to receive either DTIC (850 mg/m(2)every 28 days) plus IFN-alpha2a/b (3 MIU/m(2), twice on day 1, once daily from days 2 to 5; 5 MIU/m(2)3 times a week from week 2 to 4) with or without IL-2 (4.5 MIU/m(2)for 3 hours i.v. on day 3; 9.0 MIU/m(2) i.v. day 3/4; 4.5 MIU/m(2) s.c. days 4 to 7). The treatment plan required at least 2 treatment cycles (8 weeks of therapy) for every patient. Of 290 randomized patients 281 were eligible for an intention-to-treat analysis. There was no difference in terms of survival time from treatment onset between the two arms (median 11.0 months each). In 273 patients treated according to protocol tumour response was assessable. The response rates did not differ between both arms (P = 0.87) with 18.0% objective responses (9.7% PR; 8.3% CR) for DTIC plus IFN-alpha as compared to 16.1% (8.8% PR; 7.3% CR) for DTIC, IFN-alpha and IL-2. Treatment cessation due to adverse reactions was significantly more common in patients receiving IL-2 (13.9%) than in patients receiving DTIC/IFN-alpha only (5.6%). In conclusion, there was neither a difference in survival time nor in tumour response rates when IL-2, applied according to the combined intravenous and subcutaneous schedule used for this study, was added to DTIC and IFN-alpha. However, toxicity was increased in melanoma patients treated with IL-2. Further phase III trials with continuous infusion and higher dosages must be performed before any final conclusions can be drawn on the potential usefulness of IL-2 in biochemotherapy of advanced melanoma. PMID:11308250

Hauschild, A; Garbe, C; Stolz, W; Ellwanger, U; Seiter, S; Dummer, R; Ugurel, S; Sebastian, G; Nashan, D; Linse, R; Achtelik, W; Mohr, P; Kaufmann, R; Fey, M; Ulrich, J; Tilgen, W



Methyl-parathion and organophosphorous pesticide metabolites modify the activation status and interleukin-2 secretion of human peripheral blood mononuclear cells.  


Organophosphorous (OP) compounds are the most commonly used pesticides. There are reports on susceptibility to the toxic effects of OP pesticides, but no information exists regarding the toxicity of their metabolites. To determine the metabolites' contribution to the OP pesticide immunotoxic effects, human peripheral blood mononuclear cells (PBMCs) were treated with the parent compound methyl-parathion (MP) and the following OP pesticide alkyl-phosphorous metabolites: diethylphosphate (DEP), diethylthiophosphate (DETP), diethyldithiophosphate (DEDTP), dimethylphosphate (DMP), and dimethyldithiophosphate (DMDTP). Activation and function of the PBMCs were examined by assessment of phytohemagglutinin (PHA)-induced proliferative response, interleukin-2 (IL-2) secretion, and CD25 and CD69 expression. Treatments with DMP, DEP, DETP and DEDTP for 48h produced significant toxicity in human PBMCs, but did not affect their proliferative response to PHA. Only MP reduced cell proliferation by 30%. DEDTP decreased the proportion of PBMCs expressing CD25. This effect was associated with a reduction of IL-2 secretion, which was also reduced by MP and DMP treatments. In contrast, DETP and DEDTP treatments increased the expression of CD69. DMP, DETP and DEDTP were more consistently involved in modulating the PBMC response to PHA. PMID:15993741

Lima, Alejandro; Vega, Libia



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

PubMed Central

Previously we described the clinical aspects of a phase I study of prolonged continuous infusion of low-dose recombinant interleukin-2 (rIL-2). In the present paper we report several immunological effects in 13 patients with melanoma and renal cell cancer treated on an out-patient basis with rIL-2 for uninterrupted periods ranging from 5 to 18 weeks. Groups of three patients were treated at following dose levels 0.18, 0.6, 1.8 or 6 x 10(6) IU m-2 24 h-1 and one patient was treated with 3 x 10(6) IU m-2 24 h-1. Prolonged rIL-2 treatment resulted in a dose-dependent and sustained increase in the percentage and absolute number of (CD56+, CD8dim) natural killer cells. Within this population a preferential increase in the CD56bright cells with low expression of CD16 was observed. The CD27 antigen was also upregulated in the CD56bright CD16dim population. This increase of NK cells was accompanied by an enhancement of the cytotoxic capacity of the peripheral lymphocytes. No consistent signs of T cell activation or expansion were noted.

Vlasveld, L. T.; Hekman, A.; Vyth-Dreese, F. A.; Rankin, E. M.; Scharenberg, J. G.; Voordouw, A. C.; Sein, J. J.; Dellemijn, T. A.; Rodenhuis, S.; Melief, C. J.



Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes.  

PubMed Central

The proliferating-cell nuclear antigen (PCNA) gene encodes an auxiliary factor of DNA polymerase delta and functions in DNA replication during S phase. It is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the regulatory role of the 5'-flanking sequence of the murine PCNA gene in interleukin 2 (IL-2)-responsive cloned T cells (L2). Analysis of a set of deletion constructs in transient transfection assays measuring heterologous reporter gene (luciferase) activity demonstrated that the 182-bp 5'-flanking region provides full promoter activity in IL-2-stimulated L2 cells. While many elements contribute to PCNA promoter strength in IL-2-stimulated cells, the largest decrease in activity occurred with deletion of the tandem CRE (cyclic AMP response element) binding sites located at nucleotides -37 to -52. With a gel mobility shift assay, several IL-2-inducible DNA-protein complexes were detected, including CREB (CRE-binding) and ATF1 (activating transcription factor) proteins that are specific for the PCNA-CRE sequence. Methylation interference analysis confirmed specific binding of these proteins to the CRE sites. Mutation at the PCNA-CRE motif abolishes IL-2-inducible binding and reduces substantially PCNA promoter activity. These results indicate that IL-2-stimulated PCNA transcription may be partially mediated by these CRE-binding proteins. Images

Huang, D; Shipman-Appasamy, P M; Orten, D J; Hinrichs, S H; Prystowsky, M B



Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: I. Phenotypic and functional analysis of the peripheral blood mononuclear cells.  


The efficacy of recombinant interleukin-2 (rIL-2) or rIL-2 plus lymphokine-activated killer (LAK) cells in cancer therapy has been demonstrated by several groups both in experimental models in animals and clinical trials in humans, but their effects in vivo have yet to be clarified. Starting February 1988, we have treated 12 patients affected by advanced renal cancer with rIL-2 + LAK cells according to an open, non-randomized, phase II trial. Immediately before each rIL-2 infusion and during the last day of infusion, immunological tests were performed on the patients' peripheral blood mononuclear cells. During rIL-2 infusion we have observed a slight increase of the spontaneous cell proliferation and of natural killer (NK) and LAK activity; phenotypic analysis showed a significant decrease in the CD4+ T-lymphocyte subset, both in percentage and in absolute number. Conversely, before each cycle CD4+ cells increased when compared to basal values. No significant variations were observed in the CD8+ T-lymphocyte subset. Furthermore, a significant increase of the NK cells (CD3- CD56+ CD16+) was evident during rIL-2 infusion. PMID:2289209

Fortis, C; Ferrero, E; Besana, C; Biffi, M; Heltai, S; Galli, L; Borri, A; Schoenheit, A; Rugarli, C



Prophylactic immunosuppression with anti-interleukin-2 receptor monoclonal antibody LO-Tact-1 versus OKT3 in liver allografting. A two-year follow-up study.  


A prospective trial was conducted in 129 recipients of primary liver transplantation, to compare induction immunosuppression using triple drug therapy (cyclosporine, steroids, and azathioprine; group 1, n = 42), versus triple drug therapy with a 10-day course of OKT3 (group 2, n = 44) or of the anti-interleukin-2 receptor monoclonal antibody LO-Tact-1 (group 3, n = 43). Two-year actual patient survival rates were 64%, 79%, and 93% in groups 1, 2, and 3, respectively (1 vs. 2, NS; I vs. III, P = 0.003; 2 vs. 3, NS). Up to 2 years after transplantation, 18%, 44%, and 53% of the grafts in groups 1, 2, and 3, respectively, had not experienced steroid-resistant acute rejection (1 vs. 2, P = 0.002; 1 vs. 3, P = 0.007; 2 vs. 3, NS). The overall incidence of chronic rejection was 4%. OKT3 therapy, but not LO-Tact-1, significantly increased the incidence of cytomegalovirus infections (P = 0.019). In conclusion, immunoprophylaxis with LO-Tact-1 seemed to provide a liver graft acceptance rate at least as satisfactory as that with OKT3, without an increase in the incidence of infections. PMID:8629306

Reding, R; Feyaerts, A; Vraux, H; Latinne, D; De La Parra, B; Cornet, A; Cormont, F; Jamart, J; Sokal, E; de Ville de Goyet, J; Lerut, J; Bazin, H; Otte, J B



A 100-kilodalton protein is associated with the murine interleukin 2 receptor: Biochemical evidence that p100 is distinct from the. alpha. and. beta. chains  

SciTech Connect

Two proteins that specifically bind the T-cell growth factor interleukin 2 (IL-2) have been identified previously on the surface of T cells; these proteins have been designated IL-2R{alpha} and IL-2R{beta} for the {alpha} and {beta} chains of the IL-2 receptor (IL-2R). The association of these independent binding proteins with each other on the surface of activated T cells correlates with the generation of high-affinity binding sites. These high-affinity sites transduce the major mitogenic signal of IL-2, yet the mechanisms of association of the {alpha} and {beta} chains with each other as well as signal transduction in response to IL-2 are unknown. Cotransfection experiments of cDNAs encoding the {alpha} and {beta} chains in T cells and fibroblasts have suggested functional requirements for other T cell-specific factor(s). The authors now provide biochemical evidence for a distinct 100-kDa protein that interacts with the {alpha} or {beta} chains, or both, on the surface of the IL-2-dependent cell line CTLL-2 as well as activated murine splenocytes. This same 100-kDa protein is capable of being chemically cross-linked to {sup 125}I-labeled IL-2.

Sharon, M.; Gnarra, J.R.; Leonard, W.J. (National Institutes of Health, Bethesda, MD (USA))



Selective unresponsiveness to beta cell autoantigens after induction immunosuppression in pancreas transplantation with anti-interleukin-2 receptor antibody versus anti-thymocyte globulin  

PubMed Central

Pancreas transplantation in type 1 diabetes patients could result in (re)activation of allo- and autoreactive T lymphocytes. Anti-thymocyte globulin (ATG) induction treatment is a successful, but broadly reactive anti-lymphocyte therapy used in pancreas and islet transplantation. A more selective alternative is daclizumab, a monoclonal antibody directed against the interleukin-2 receptor (CD25) on activated lymphocytes. We tested the hypothesis that daclizumab is more selective and has less immunological side effects than ATG. Thirty-nine simultaneous pancreas–kidney transplantation patients with type 1 diabetes were randomized for induction therapy with ATG or daclizumab. Auto- and recall immunity was measured cross-sectionally by lymphocyte stimulation tests with a series of auto- and recall antigens in 35 successfully transplanted patients. T cell autoimmunity to islets was low in both groups, except for a marginal but significantly higher reactivity against glutamic acid decarboxylase (GAD)65 in daclizumab-treated patients. The memory responses to recall antigens were significantly higher in the daclizumab-treated group compared to ATG-treated patients, specifically against purified protein derivative (PPD) (anti-bacterial immunity), Haemophilus influenzae virus matrix protein-1 (anti-viral immunity) and p53 [anti-tumour (auto)immunity]. These data imply that daclizumab is more specifically affecting diabetes-related immune responses than ATG. The autoimmunity is affected effectively after daclizumab induction, while memory responses towards bacterial, viral and tumour antigens are preserved.

van de Linde, P; Boog, P J M vd; Tysma, O M H; Elliott, J F; Roelen, D L; Claas, F H J; de Fijter, J W; Roep, B O



Novel CD28-Responsive Enhancer Activated by CREB/ATF and AP-1 Families in the Human Interleukin-2 Receptor ?-Chain Locus  

PubMed Central

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) critically regulates the T-cell immune response, and the ? chain CD25/IL-2R? is required for the formation of the high-affinity receptor. Tissue-specific, inducible expression of the IL-2R? gene is regulated by at least three positive regulatory regions (PRRI, PRRII, and PRRIII), but none responded to CD28 engagement in gene reporter assays although CD28 costimulation strongly amplifies IL-2R? gene transcription. By DNase I hypersensitivity analysis, we have identified a novel TCR-CD3- and CD28-responsive enhancer (CD28rE) located 8.5 kb 5? of the IL-2R? gene. PRRIV/CD28rE contains a functional CRE/TRE element required for CD28 signaling. The T-cell-specific, CD28-responsive expression of the IL-2R? gene appears controlled through PRRIV/CD28rE by cooperation of CREB/ATF and AP-1 family transcription factors.

Yeh, Jung-Hua; Lecine, Patrick; Nunes, Jacques A.; Spicuglia, Salvatore; Ferrier, Pierre; Olive, Daniel; Imbert, Jean



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

SciTech Connect

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.

Zu, Youli; Shigesada, Katsuya; Hanaoka, Masao; Namba, Yuziro (Kyoto Univ. (Japan)); Nishida, Eisuke (Univ. of Tokyo (Japan)); Kubota, Ichiro (Suntory Bio-Pharma Tech Center, Gunma (Japan)); Kohno, Michiaki (Gifu Pharmaceutical Univ., (Japan))



Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells.  


Listeria monocytogenes infection generates T helper 1 (Th1) effector memory cells and CC chemokine receptor 7 (CCR7)(+) cells resembling central memory cells. We tracked endogenous L. monocytogenes-specific CD4(+) T cells to determine how these memory cells are formed. Two effector cell populations were already present several days after infection. One highly expressed the T-bet transcription factor and produced Th1 memory cells in an interleukin-2 (IL-2) receptor-dependent fashion. The other resided in the T cell areas, expressed CCR7 and CXC chemokine receptor 5 (CXCR5), and like follicular helper cells depended on the Bcl6 transcription factor and inducible costimulator ligand on B cells. The CCR7(+)CXCR5(+) effector cells produced similar memory cells that generated diverse effector cell populations in a secondary response. Thus, Th1 effector memory and follicular helper-like central memory cells are produced from early effector cell populations that diverge in response to signals from the IL-2 receptor, Bcl6, and B cells. PMID:22018468

Pepper, Marion; Pagán, Antonio J; Igyártó, Botond Z; Taylor, Justin J; Jenkins, Marc K



Enhancement of the immunogenicity of an alphavirus replicon-based DNA vaccine against classical swine fever by electroporation and coinjection with a plasmid expressing porcine interleukin 2.  


Alphavirus replicon-based DNA vaccines have emerged as a promising approach to generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for other approaches to enhance the vaccine potency. In this study, electroporation (EP) and a plasmid expressing porcine interleukin 2 (IL-2) were used to improve the immunogenicity of an alphavirus replicon-based DNA vaccine pSFV1CS-E2 against classical swine fever (CSF). Pigs were immunized with pSFV1CS-E2 alone or together with IL-2 by EP or by simple intramuscular injection. The results showed that EP combined with IL-2 resulted in marked enhancement of E2-specific antibody responses. Moreover, CSFV-specific lymphocyte proliferation, IFN-? and IL-4 responses were increased significantly in the pSFV1CS-E2+IL-2/EP group. Pigs immunized with pSFV1CS-E2 plus IL-2 by EP were completely protected from lethal challenge, which is comparable to the sterilizing immunity and full protection offered by the live attenuated vaccine C-strain and in contrast with the incomplete protection conferred by pSFV1CS-E2 without or with IL-2 or EP alone, as demonstrated by the presence of pathological changes or/and viral loads. We conclude that EP in combination with IL-2 can significantly improve the immunogenicity of the plasmid DNA vaccine. PMID:22469861

Tian, Da-Yong; Sun, Yuan; Wai, Sing Fai; Lee, Fuk Ki; Meng, Qi-Lin; Suen, Kar Man; Wang, Nan; Han, Wen; Li, Su; Li, Yong-Feng; Li, Dan; Ling, Li-Jun; Liao, Ya-Jin; Qiu, Hua-Ji



The synthetic peptide related to the central part of human interleukin-2 molecule accelerates growth and vascularization of sarcoma 180 in mice.  


The synthetic peptide C-1-6 related to the central part of human interleukin 2 molecule (sequence 59-72; N- and C-modified) had been shown previously to inhibit cytotoxic activity of macrophages converting them to synthesis of growth factors. In this paper the effect of C-1-6 on growth of sarcoma 180 in mice was studied. C-1-6 significantly accelerated tumor growth having been injected into mice in dose 5 or 50 microg per animal since the 4th day after tumor cells transplantation. Supernatants of Mphi in vitro activated by C-1-6 (10 microg/ml) and injected into mice also accelerated significantly sarcoma mass diurnal increasing as compared to mice treated with supernatants of non-activated Mphi or activated with bacterial lipopolysaccharide. A single injection of C-1-6 into mice either at the day or at the next day of tumor cells inoculation increased significantly the number of vessels growing up to transplant, thus the forming of the vascular bed had preceded tumor volume enlargement. PMID:10965981

Okulov, V B; Ushmorov, A G; Voytenkov, B O; Onoprienko, L V; Mikhaleva, I I; Ivanov, V T



Antitumor Efficacy of Intravesical BCG, Gemcitabine, Interferon-? and Interleukin-2 as Mono- or Combination-Therapy for Bladder Cancer in an Orthotopic Tumor Model  

PubMed Central

Objective: To reduce adverse effects and improve efficacy of intravesical BCG for bladder cancer, alternative treatment options were investigated in an orthotopic rat tumor model. Methods: Superficial bladder cancer was established in syngeneic female rat bladders by instillation of AY-27 cells. Animals were randomly assigned to treatment groups including dose escalation of intravesical BCG with or without interferon-? (IFN-?) or interleukin-2 (IL-2); or graded doses of gemcitabine alone; or BCG plus gemcitabine. Treatments were given twice weekly for 3 weeks. Rats in control groups received saline instillations. Treatment response was monitored by animals’ well-being, survival days, tumor growth inhibition, and histological examination at necropsy. Results: Rats receiving monotherapy with intravesical BCG, gemcitabine, or IFN-?, attained significantly better survival and tumor reduction compared with control (P = 0.002; 0.001; 0.002, respectively, Log-rank Test). A dose-dependent treatment response was observed in animals with established bladder tumor receiving escalated BCG instillations. Only high-dose BCG significantly improved animal survival. Although high-dose BCG plus gemcitabine or IFN-? did not increase benefit over monotherapies, low-dose BCG plus IL-2 did show improved efficacy (P = 0.01). Conclusion: Intravesical monotherapies with gemcitabine and IFN-? were as effective as BCG for treatment of early non-muscle-invasive urothelial bladder cancer in this immune competent rat model. Combining these agents with high-dose BCG did not further increase efficacy. However, combining low-dose BCG with IL-2 enhanced BCG effectiveness.

Xiao, Zhengwen; Hanel, Erich; Mak, Allan; Moore, Ronald B.



Intranasal administration of human immunodeficiency virus type-1 (HIV-1) DNA vaccine with interleukin-2 expression plasmid enhances cell-mediated immunity against HIV-1.  

PubMed Central

DNA vaccine against human immunodeficiency virus type-1 (HIV-1) can induce substantial levels of HIV-1-specific humoral and cell-mediated immunity. To develop more potent HIV-1 DNA vaccine formulations, we used a murine model to explore the immunomodulatory effects of an interleukin-2 (IL-2) expression plasmid on an HIV-1 DNA vaccine following intranasal administration of the combination. When the vaccine and expression plasmid were incorporated into cationic liposomes and administered to mice, the HIV-1-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were significantly increased. Restimulated immune lymphoid cells showed enhanced production of both IL-2 and interferon-gamma and reduced secretion of IL-4. The level of total antibody to HIV-1 antigen was not greatly changed by coadministration of the DNA vaccine and IL-2 expression plasmid. An analysis of serum HIV-1-specific IgG subclasses showed a significant drop in the IgG1/IgG2a ratio in the group that received the plasmid-vaccine combination. These results demonstrate that the IL-2 expression plasmid strongly enhances the HIV-1-specific immune response via activation of T helper type-1 cells. Images Figure 1 Figure 2

Xin, K Q; Hamajima, K; Sasaki, S; Honsho, A; Tsuji, T; Ishii, N; Cao, X R; Lu, Y; Fukushima, J; Shapshak, P; Kawamoto, S; Okuda, K



PMA and interleukin-1 do not necessarily perform the same function in the induction of IL-2 secretion and interleukin-2 receptor expression  

SciTech Connect

The murine T-lymphoma cell line EL-4 was used to investigate the relative roles of interleukin-1 (IL-1), calcium, and activators of protein kinase C (PKC) in the induction of interleukin-2 (IL-2) secretion. EL-4 cells produced maximal levels of IL-2 in response to stimulation with 20 nM (but not 1 nM) phorbol myristate acetate (PMA). IL-1 and 1 nM PMA each synergized with calcium ionophore A23187, producing significant but submaximal levels of IL-2. Furthermore, an inhibitor of PKC inhibited IL-2 secretion stimulated by IL-1 and A23187. These results suggest that IL-1 and PMA both activate PKC in a similar manner. However, additional experiments indicated that IL-1 and PMA may have distinct mechanisms of promoting IL-2 production. IL-1 increased IL-2 production (and IL-2 receptor expression) induced by 1 nM PMA, 50 diacylglycerol, or combinations of either of these with A23187 to maximal levels. The synergy exhibited by IL-1 and low doses of PMA indicates that IL-1 and PMA may act at separate sites in the biochemical pathway, but high doses of PMA (20 nM) may replace the function of IL-1 as well.

Simon, P.L.; Truneh, A.



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

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.

MacDonald, H L; Neway, J O



Reversal of Human Immunodeficiency Virus Type 1 Protein-Induced Inhibition of Natural Killer Cell Activity by Alpha Interferon and Interleukin-2  

PubMed Central

A recombinant fusion peptide, Env-Gag, derived from the human immunodeficiency virus type 1 (HIV-1) genome corresponding to a defined portion of the envelope (Env) and internal core (Gag) proteins was examined for immunoregulatory effects on the cytotoxic activity of natural killer (NK) cell-enriched, large granular lymphocytes (LGL) from healthy donors. Percoll-separated, NK cell-enriched LGL precultured for 24 h with Env-Gag at 10- and 50-ng/ml concentrations, which significantly stimulated lymphocyte proliferation, caused significant suppression of NK cell activity. Denatured Env-Gag did not cause any effect on the NK cell activity of LGL. Two other control peptides, one derived from the Escherichia coli vector used to clone the HIV Env-Gag fusion peptide and the other derived from a non-HIV-1 viral antigen (rubeola virus), did not produce any observable effect on the NK cell activity of LGL, demonstrating the specificity of the effect produced by Env-Gag. Subsequent treatment of LGL with alpha interferon (IFN-?) or interleukin 2 (IL-2) alone partially reversed the Env-Gag-induced suppression of NK cell activity. However, LGL treated with both IFN-? and IL-2 completely reversed the suppression of NK cell cytotoxicity by Env-Gag. The combined effect of IFN-? and IL-2 in enhancing NK cell activity may provide a novel therapeutic approach to the restoration of depressed NK cell activity observed in HIV-infected patients.

Nair, Madhavan P. N.; Schwartz, Stanley A.



Reversal of human immunodeficiency virus type 1 protein-induced inhibition of natural killer cell activity by alpha interferon and interleukin-2.  


A recombinant fusion peptide, Env-Gag, derived from the human immunodeficiency virus type 1 (HIV-1) genome corresponding to a defined portion of the envelope (Env) and internal core (Gag) proteins was examined for immunoregulatory effects on the cytotoxic activity of natural killer (NK) cell-enriched, large granular lymphocytes (LGL) from healthy donors. Percoll-separated, NK cell-enriched LGL precultured for 24 h with Env-Gag at 10- and 50-ng/ml concentrations, which significantly stimulated lymphocyte proliferation, caused significant suppression of NK cell activity. Denatured Env-Gag did not cause any effect on the NK cell activity of LGL. Two other control peptides, one derived from the Escherichia coli vector used to clone the HIV Env-Gag fusion peptide and the other derived from a non-HIV-1 viral antigen (rubeola virus), did not produce any observable effect on the NK cell activity of LGL, demonstrating the specificity of the effect produced by Env-Gag. Subsequent treatment of LGL with alpha interferon (IFN-alpha) or interleukin 2 (IL-2) alone partially reversed the Env-Gag-induced suppression of NK cell activity. However, LGL treated with both IFN-alpha and IL-2 completely reversed the suppression of NK cell cytotoxicity by Env-Gag. The combined effect of IFN-alpha and IL-2 in enhancing NK cell activity may provide a novel therapeutic approach to the restoration of depressed NK cell activity observed in HIV-infected patients. PMID:10618286

Nair, M P; Schwartz, S A



Effect of recombinant human interleukin-2 on the course of experimental chronic respiratory tract infection caused by Klebsiella pneumoniae in mice.  

PubMed Central

The effect of recombinant human interleukin-2 (rIL-2) on the course of experimental chronic respiratory tract infection caused by Klebsiella pneumoniae in mice was examined. rIL-2 was administered subcutaneously once a day for 7 or 14 days, starting 2 weeks after the mice were infected. Administration of 2 or 20 micrograms of rIL-2 per mouse daily for 7 days reduced bacterial counts in the lungs dose dependently. At a dose of 0.2 microgram per day, proliferation of bacteria in the lungs was suppressed after 14 days of administration. Agglutinin titers in serum were not affected by rIL-2 treatment. Monocyte and lymphocyte counts in peripheral blood were increased by administration of 20 micrograms of rIL-2 daily for 14 days but not by treatment for 7 days. In addition, clearance of bacteria from the lungs after aerosol exposure was enhanced by treatment for 7 days before infection. Thus, rIL-2 acted therapeutically or prophylactically in the presence or absence, respectively, of a specific antigen. These effects were not abolished by anti-asialo GM1 antibody. This suggests that activation of natural killer cells does not play a critical role in the therapeutic and prophylactic effects of rIL-2.

Iizawa, Y; Nishi, T; Kondo, M; Tsuchiya, K; Imada, A



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

PubMed Central

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

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



Interaction of various cytokines with interleukin 2 in the generation of killer cells from human bone marrow: application in purging of leukemia.  


We have shown that incubation of bone marrow (BM) with interleukin 2 (IL-2) generates activated bone marrow cells (ABM) with potent tumoricidal activity in vitro and in vivo. The present study was carried out to define the interaction of other cytokines with IL-2 in generation of ABM. Our data show that interleukin 1 (IL-1), interferon (IFN)- both gamma and alpha, and tumor necrosis factor (TNF-alpha) significantly increased the cytolytic potential of ABM. Interleukin 3, interleukin 4, transforming growth factor-beta and adherent cells were reduced, while granulocyte-macrophage colony-stimulating factor had no influence on the generation of cytolytic activity. IL-1 was enhanced while TNF-alpha depressed the BM progenitor cell activity in vitro. The IL-2-induced purging ability of BM contaminated with leukemic cells was increased by IL-1, TNF-alpha and IFN-gamma. This study shows that biomodulation of BM with combination of cytokines in vitro can be useful in purging a large leukemic burden. PMID:1921458

Charak, B S; Agah, R; Gray, D; Mazumder, A



A Phase II Study of Bevacizumab and High-dose Interleukin-2 in Patients With Metastatic Renal Cell Carcinoma: A Cytokine Working Group (CWG) Study.  


Overexpression of vascular endothelial growth factor in renal cell carcinoma (RCC) leads to angiogenesis, tumor progression, and inhibition of immune function. We conducted the first phase II study to estimate the efficacy and safety of bevacizumab with high-dose interleukin-2 (IL-2) therapy in patients with metastatic RCC. Eligible patients had predominantly clear cell metastatic RCC, measurable disease, a Karnofsky Performance Status of ?80%, and adequate end-organ function. IL-2 (600,000 IU/kg) was infused intravenously every 8 hours (maximum 28 doses) during two 5-day cycles on days 1 and 15 of each 84-day course. Bevacizumab (10 mg/kg) was infused intravenously every 2 weeks beginning 2 weeks before initiating IL-2. Fifty of 51 eligible patients from 8 centers were enrolled. Median progression-free survival (PFS) was 11.2 months (90% confidence interval, 5.7-17.7), and 2-year PFS was 18% (90% confidence interval, 8%-27%). Responses included 4 complete (8%) and 11 partial (22%) responses. Toxicities did not exceed those expected from each agent alone. Combining IL-2 plus bevacizumab is feasible, with a response rate and PFS at least as high as reported previously for the single agents. The regimen did not appear to enhance the rate of durable major responses over that of IL-2 alone. PMID:24145360

Dandamudi, Uday B; Ghebremichael, Musie; Sosman, Jeffrey A; Clark, Joseph I; McDermott, David F; Atkins, Michael B; Dutcher, Janice P; Urba, Walter J; Regan, Meredith M; Puzanov, Igor; Crocenzi, Todd S; Curti, Brendan D; Vaishampayan, Ulka N; Crosby, Nancy A; Margolin, Kim A; Ernstoff, Marc S


Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures  

PubMed Central

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis–trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis–trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the ?-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively.

Joseph, Raji E.; Ginder, Nathaniel D.; Hoy, Julie A.; Nix, Jay C.; Fulton, D. Bruce; Honzatko, Richard B.; Andreotti, Amy H.



Functional dissection of p56lck, a protein tyrosine kinase which mediates interleukin-2-induced activation of the c-fos gene.  

PubMed Central

Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation. Images

Shibuya, H; Kohu, K; Yamada, K; Barsoumian, E L; Perlmutter, R M; Taniguchi, T



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


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

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



JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport  

Microsoft Academic Search

Expansion of the polyglutamine (polyQ) stretch in the androgen receptor (AR) protein leads to spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease characterized by lower motor neuron degeneration. The pathogenic mechanisms underlying SBMA remain unknown, but recent experiments show that inhibition of fast axonal transport (FAT) by polyQ-expanded proteins, including polyQ-AR, represents a new cytoplasmic pathogenic lesion. Using pharmacological,

Gerardo Morfini; Gustavo Pigino; Györgyi Szebenyi; Yimei You; Sarah Pollema; Scott T Brady



Eosinophil peroxidase catalyzes JNK-mediated membrane blebbing in a Rho kinase-dependent manner  

Microsoft Academic Search

Eosinophilic influx is characteristic of numerous inflammatory conditions. Eosinophil peroxidase (EPO) is a major enzyme present in eosinophils and upon degranulation, becomes re- leased into the airways of asthmatics. As a result of its cationic nature and its ability to catalyze the formation of highly toxic oxidants, EPO has signif- icant potential to induce cellular injury. The focus of the

Brian McElhinney; Matthew E. Poynter; Punya Shrivastava; Stanley L. Hazen; Yvonne M. W. Janssen-Heininger



Inhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation  

Microsoft Academic Search

The aim of this study was to investigate the mechanism of inhibition of mitochondrial aldehyde dehydrogenase (ALDH2) by carbon tetrachloride (CCl4). CCl4 administration caused marked hepatocyte ballooning and necrosis in the pericentral region. CCl4 also inhibited hepatic ALDH2 activity in a time-dependent manner without altering the protein level, suggesting ALDH2 inhibition through covalent modifications such as phosphorylation by JNK. To

Kwan-Hoon Moon; Young-Mi Lee; Byoung-Joon Song



Role of zinc and ?2macroglobulin on thymic endocrine activity and on peripheral immune efficiency (natural killer activity and interleukin 2) in cervical carcinoma  

PubMed Central

Decreased natural killer (NK) activity as well as interleukin 2 (IL-2) are risk factors for the progression of cervical carcinoma. NK activity and IL-2 may be thymus controlled. Plasma levels of active thymulin, a zinc-dependent thymic hormone (ZnFTS), are reduced in cancer because of the low peripheral zinc bioavailability. Zinc and thymulin are relevant for normal immune functions. ?2-Macroglobulin is an inhibitor of matrix metalloproteases (MMPs) against invasive tumour proliferation. Because ?2-macroglobulin has a binding affinity (Kd) for zinc that is higher than does thymulin, it may play a key role in immune efficiency in cancer. Plasma samples of 22 patients (age range 35–60 years) with locally advanced squamous cervical carcinoma and with FIGO stage Ib2–IIb were examined. They showed reduced active thymulin, decreased NK activity and IL-2 production, increased soluble IL-2 receptor (sIL-2R) and augmented ?2-macroglobulin in the circulation, whereas plasma zinc levels were within the normal range for age. Significant positive correlations were found between zinc or active thymulin and ?2-macroglobulin (r = 0.75, P< 0.01, r = 0.78, P< 0.01, respectively) in cancer patients. In vitro zinc increases IL-2 production from peripheral blood mononuclear cells (PBMCs) of cancer patients. These data suggest that an increase in ?2-macroglobulin, which competes with thymulin for zinc binding, may be involved in causing a thymulin deficit with a consequent decrease of IL-2 and NK cytotoxicity. Thus, physiological zinc treatment in cervical carcinoma maybe restores impaired central and peripheral immune efficiency. © 1999 Cancer Research Campaign

Mocchegiani, E; Ciavattini, A; Santarelli, L; Tibaldi, A; Muzzioli, M; Bonazzi, P; Giacconi, R; Fabris, N; Garzetti, G G



Interleukin 2 production in HTLV-III/LAV infection: evidence of defective antigen-induced, but normal mitogen-induced IL-2 production.  

PubMed Central

We studied mitogen- and antigen-induced interleukin 2 (IL-2) production in HTLV-III/LAV infected and non-infected individuals and compared the results with T cell subpopulations, and with mitogen- and antigen-induced DNA synthesis and production of leucocyte migration inhibitory factor (LIF) in order to understand the controversial findings related to IL-2 production in HTLV-III/LAV infection. The HTLV-III/LAV antibody positive group showed immunological defects: low T helper (Th) cells, high T-suppressor (Ts) cells, reduced mitogen- and antigen-induced DNA-synthesis, but LIF production comparable to the HTLV-III/LAV antibody negative group. The total amount of IL-2, produced either as a response to a mitogenic stimulus or as a response to a soluble antigenic (purified protein derivative of tuberculin, PPD) stimulus, was lower in the HTLV-III/LAV antibody positive group. However, adjusting the IL-2 production to the amount of Th-cells showed that the IL-2 produced by a standard number of Th-cells after mitogen induction was similar in HTLV-III/LAV infected and non-infected individuals. In contrast, the ability of Th-cells of infected persons to produce IL-2, or to proliferate as a response to a soluble antigenic stimulus, was considerably diminished. We conclude that HTLV-III infection leads to a selective incapability to mount a specific Th-cell response either due to an intrinsic defect in the Th-cell population or due to metabolic and/or functional disturbances in antigen-processing and presenting accessory cells.

Antonen, J; Krohn, K



Concanavalin A induced phosphatidyl inositol turnover in mouse thymocytes is not coupled to activation of protein kinase C or mitogenic response to interleukin 2  

SciTech Connect

The authors have examined the connection between phosphoinositide metabolism and lectin-activation of T-cells by measuring in vivo incorporation of /sup 32/PO/sub 4/ into phospholipids. Mouse thymocytes treated with Concanavalin A (Con A) do not proliferate nor develop mitogenic responsiveness to Interleukin 2 (IL-2). Co-treatment with Con A and either activated splenocyte conditioned medium (CM) or the Protein Kinase C (PK-C) activator, Phorbol Myristic Acetate (PMA), induces IL-2 responsiveness with a parallel increase in expression of IL-2 surface responsiveness with a parallel increase in expression of IL-2 surface receptors. Con A alone stimulates phosphoinositol (PI) turnover by 2.5-3.7 fold but has no effect on incorporation of /sup 32/PO/sub 4/ into other phospholipids, while PI turnover is unaffected by CM with or without Con A. Diacyl Glycerol (DG) arising from the breakdown of phosphoinositides, is postulated to activate PK-C (as is also PMA). These data suggest that PI breakdown, stimulated by Con A, does not produce sufficient DG to activate PK-C and/or PK-C activation by itself is not sufficient to activate IL-2 responsiveness and the generation of IL-2 receptors. The growth factor(s) in CM also do not appear to activate PK-C but do co-activate IL-2 responsiveness. The authors have observed that CM or PMA but not Con A treatment of thymocytes specifically stimulates the phosphorylation of a 55Kd polypeptide suggesting that other kinases with similar specificities to PK-C are activated by CM and PMA.

Feister, A.J.; Sage, H.J.



[Construction and immunogenicity of a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus and the porcine interleukin 2 in rabbits].  


To construct a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus (CSFV) and the porcine interleukin 2 (pIL-2), the CSFV E2 gene and pIL-2 gene were amplified respectively from the plasmids pMD19-T-E2 and pMD19-T-pIL-2 by PCR. E2-pIL-2 fusion gene was obtained by using 5 consecutive glycine codons as a linker and cloned into the adenoviral shuttle plasmid AdTrack. The AdTrack-E2-pIL-2 was linearized and transformed into E. coli BJ5183 with the backbone plasmid AdEasy1. The resultant recombinant plasmid AdEasy-E2-pIL-2 was transfected into the 293 cells where the recombinant adenovirus rAd-E2-pIL-2 was produced. The immunogenicity of rAd-E2-pIL-2 was evaluated in rabbits. The results of RT-PCR and Western-blotting showed that rAd-E2-pIL-2 could carry and express E2 and pIL-2 proteins. The titer of the rAd-E2-pIL-2 was 10(8.12) PFU/mL. After immunized with rAd-E2pIL-2, The injected rabbits developed a high level of CSFV specific antibodies. Regular fever was not detected in the rAd-E2-pIL-2-immunized rabbits upon challenge with CSFV C stain, and specific lymphoproliferative responses to the CSFV was detected in the lymphocytes from the immunized rabbits. In conclusion, rAd-E2-pIL-2 was constructed successfully and it could be an attractive vaccine candidate against CSFV. PMID:21043139

He, Lei; Zhang, Yan-ming; Xu, Yan-zhao; Tang, Qing-hai; Wang, Jing; Yang, Xiao-yun; Dai, Chen; Xiang, Hua; Chang, Peng-xiang; Lin, Zhi



Systemic interleukin-1 alpha and interleukin-2 secretion in response to acute stress and to corticotropin-releasing hormone in humans.  


Acute stress results in activation of the hypothalamic-pituitary-adrenal (HPA) axis. ACTH and cortisol secretion is stimulated by corticotropin-releasing hormone (CRH). It has also been shown that activation of the HPA axis during stress is accompanied by changes in the immune response. However, little is known about the influence of acute stress on the release of cytokines such as interleukin-1 (IL-1) or interleukin-2 (IL-2). In this study, we determined serum IL-1 alpha and IL-2 levels in 19 patients undergoing the acute stress of angioplasty for coronary artery disease. A second protocol was devised to determine serum IL-1 alpha and IL-2 concentrations as well as lymphocyte subpopulations in 10 normal volunteers receiving 1 microgram kg-1 human CRH intravenously. Finally, IL-1 alpha concentrations were measured in CRH-incubated mononuclear cell (MNC) and monocyte cultures. In response to the stress of angioplasty, ACTH and cortisol as well as IL-1 alpha and IL-2 concentrations were clearly above baseline levels (IL-1 alpha, mean +/- SEM, baseline: 1.39 +/- 0.34 ng ml-1, after angioplasty: 2.64 +/- 0.73 ng ml-1, P < 0.05; IL-2, baseline: 1.2 +/- 0.13 ng ml-1, after angioplasty: 2.8 +/- 1.14 ng ml, P < 0.05). A similar pattern was obtained in normal subjects in response to CRH (Il-1 alpha, baseline: 0.8 +/- 0.2 ng ml-1, after angioplasty: 3.7 +/- 1.4 ng ml-1, P < 0.05; IL-2, baseline: 1.9 +/- 0.4 ng ml-1, after angioplasty: 5.4 +/- 2.2 ng ml-1, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7890016

Schulte, H M; Bamberger, C M; Elsen, H; Herrmann, G; Bamberger, A M; Barth, J



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

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.

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



Structure-function relationships for the interleukin 2 receptor: location of ligand and antibody binding sites on the Tac receptor chain by mutational analysis.  


The Tac protein plays a role in high- and low-affinity interleukin 2 (IL-2) receptors. A mutational survey of this molecule identified several small segments in which the binding of IL-2 was particularly sensitive to amino acid substitutions. Two of the segments (residues 1-6 and 35-43) located in the exon 2-encoded region of the molecule overlapped the apparent binding sites of three monoclonal antibodies (anti-Tac, GL439, and H31) that block high- and low-affinity Tac-IL-2 interactions, thus supporting the hypothesis that these segments of the protein are at or near sites of receptor-ligand contact. In contrast, the apparent binding sites of antibodies (Hiei and H47) that selectively inhibit high-affinity IL-2 binding were mapped to a distinct location (residues 158-160) within the region encoded by exon 4 of the Tac gene. Since high-affinity receptors consist of a heterodimer of Tac and a second ligand-binding protein (p70), this portion of the Tac molecule may be involved in the interaction between the two receptor subunits. As expected, the binding sites of noninhibitory antibodies (7G7/B6, residues 140-144; H48, residues 170-211) did not overlap those segments in which IL-2-binding mutants were observed. These results provide a preliminary correlation of structure and function for the Tac protein that should prove useful in evaluating detailed models of the IL-2-receptor complex. PMID:3135551

Robb, R J; Rusk, C M; Neeper, M P



Therapeutic effects of idiotype vaccination can be enhanced by the combination of granulocyte-macrophage colony-stimulating factor and interleukin 2 in a myeloma model.  


Idiotype (Id) vaccination provides an interesting immunotherapeutic strategy against B-cell lymphomas. In multiple myeloma (MM), however, the therapeutic efficacy of Id vaccination has been disappointing. In an attempt to improve the antitumoral potential, we added granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 2 (IL-2) to the protocol. Balb/c mice were inoculated i.p. (d 2) with different doses (1-5 x 10(5)) of HOPC myeloma cells secreting the Ig(HOPC) Id protein. Two days later, animals were injected with 10,000 U GM-CSF i.p. for 6 d consecutively (d 0-5). On d 5 and 11, myeloma-specific immunoglobulin (Ig(HOPC)) was administered i.p. together with incomplete Freund adjuvans followed by IL-2 (2 x 10,000 U/d; i.p) for 10 d (d 5-14). In animals inoculated with 10(5) myeloma cells, treatment with IL-2 given as a single agent prolonged the median survival time (MST, 67 d) when compared with the tumour control group (MST 48 d), whereas GM-CSF did not elicit any survival benefit (MST 49 d). Complete tumour rejection could be achieved in 27% (4/15) by the combination of Id vaccination and GM-CSF. Additional treatment with IL-2 further increased antimyeloma activity. In this case, 59% of the animals showed no signs of tumour recurrence. In mice with high tumour burden (5 x 10(5)), no treatment modality achieved long-term survivors. Both natural killer (NK) cells and CD8+ T cells may be involved in the anti-tumoural immune response. These data provide evidence for the combined use of GM-CSF and IL-2 to enhance the therapeutic effectiveness of clinical cancer vaccination protocols. PMID:12492573

Stritzke, Jan; Zunkel, Tim; Steinmann, Jörg; Schmitz, Norbert; Uharek, Lutz; Zeis, Matthias



Activation of natural killer cells with interleukin 2 (IL-2) and IL-12 increases perforin binding and subsequent lysis of tumour cells.  


Natural killer (NK) cells can lyse a variety of different tumour cells by exocytosis of perforin, subsequent binding of perforin to the target cell membrane and formation of lytic pores. Some tumour cells, however, are resistant to cellular cytotoxicity. Using the NK-resistant tumour cell lines ML-2, MONOMAC-1, RPMI and L540Cy, we demonstrated that activation of NK cells with interleukin 2 (IL-2) and IL-12 resulted in significant lysis of these tumour targets. To investigate the underlying mechanisms, we isolated the cytotoxic granules from non-activated and IL-2-/IL-12-activated NK cells and compared the killing of K562 leukaemia cells (sensitive to NK cell-mediated lysis) and ML-2 leukaemia cells (resistant to NK cell-mediated lysis). In contrast to K562 cells, which were easily killed by NK-cell granules, ML-2 cells were resistant to granules from non-activated NK cells. However, granules from NK cells activated with IL-2 and IL-12 were able to induce significant tumour cell lysis. Cell death of both K562 and ML-2 cells by granules from activated NK cells was completely blocked by anti-perforin antibodies, indicating that perforin mainly accounts for the lysis induced by NK granules. Comparing granules from non-activated and IL-2-/IL-12-activated NK cells, the increased cell death of ML-2 cells was caused by an improved binding of perforin to the target cell membrane. Functional assays, however, indicated that the differences in perforin binding were not as a result of an augmented production of perforin by activated NK cells. We conclude that activation of NK cells results in an increased binding of perforin and subsequent lysis of tumour cells. PMID:11552995

Lehmann, C; Zeis, M; Uharek, L



Enhanced antitumoral effectiveness of idiotype vaccination induced by the administration of Flt3 ligand combined with interleukin 2 against a murine myeloma.  


Idiotype (Id) vaccination provides an innovative treatment modality against B-cell malignancies. In multiple myeloma patients, however, the antitumoral potential of this immunotherapeutic concept is limited. In an attempt to improve the therapeutic effectiveness of Id vaccination, we added Flt3 ligand (Flt3-L) and interleukin 2 (IL-2) to the protocol. Balb/c mice were inoculated i.p. (d -2) with different doses (1-5 x 10(5)) of HOPC myeloma cells, secreting the IgHOPC Id-protein. Two days later, animals were treated with Flt3-L (10 microg per mouse/d, given i.p) for 10 consecutive days (d 0-9). On d 5 and d 11, myeloma-specific immunoglobulin (Ig(HOPC)) was administered s.c., together with incomplete Freund adjuvans (IFA) followed by the administration of IL-2 (2 x 10.000/d given i.p) for 10 d (d 5-14). Whereas Ig(HOPC), Flt3-L or IL-2, given alone, did not elicit long-term survival, the combination of IL-2 or Flt3-L with Id vaccination achieved a complete tumour rejection in 27% and 41% of mice respectively. However, the most powerful antimyeloma effects were induced by Flt3-L + Id vaccination + IL-2: 81% of the treated animals experienced long-term survival (> 180 d). Both natural killer (NK) cells and CD8+ T cells may be involved in the antitumoral immune response. These data suggest that the combination of Flt3-L and IL-2 can be used to enhance the therapeutic effectiveness of clinical cancer vaccination protocols. PMID:11918538

Zeis, Matthias; Zunkel, Tim; Steinmann, Jörg; Schmitz, Norbert; Uharek, Lutz



Phase II trial of Modified Vaccinia Ankara (MVA) virus expressing 5T4 and high dose Interleukin-2 (IL-2) in patients with metastatic renal cell carcinoma  

PubMed Central

Background Interleukin-2 (IL-2) induces durable objective responses in a small cohort of patients with metastatic renal cell carcinoma (RCC) but the antigen(s) responsible for tumor rejection are not known. 5T4 is a non-secreted membrane glycoprotein expressed on clear cell and papillary RCCs. A modified vaccinia virus Ankara (MVA) encoding 5T4 was tested in combination with high-dose IL-2 to determine the safety, objective response rate and effect on humoral and cell-mediated immunity. Methods 25 patients with metastatic RCC who qualified for IL-2 were eligible and received three immunizations every three weeks followed by IL-2 (600,000 IU/kg) after the second and third vaccinations. Blood was collected for analysis of humoral, effector and regulatory T cell responses. Results There were no serious vaccine-related adverse events. While no objective responses were observed, three patients (12%) were rendered disease-free after nephrectomy or resection of residual metastatic disease. Twelve patients (48%) had stable disease which was associated with improved median overall survival compared to patients with progressive disease (not reached vs. 28 months, p = 0.0261). All patients developed 5T4-specific antibody responses and 13 patients had an increase in 5T4-specific T cell responses. Although the baseline frequency of Tregs was elevated in all patients, those with stable disease showed a trend toward increased effector CD8+ T cells and a decrease in Tregs. Conclusion Vaccination with MVA-5T4 did not improve objective response rates of IL-2 therapy but did result in stable disease associated with an increase in the ratio of 5T4-specific effector to regulatory T cells in selected patients. Trial registration number ISRCTN83977250

Kaufman, Howard L; Taback, Bret; Sherman, William; Kim, Dae Won; Shingler, William H; Moroziewicz, Dorota; DeRaffele, Gail; Mitcham, Josephine; Carroll, Miles W; Harrop, Richard; Naylor, Stuart; Kim-Schulze, Seunghee



Human Leukocyte Antigen and Interleukin 2, 10 and 12p40 Cytokine Responses to Measles: Is There Evidence of the HLA Effect?  

PubMed Central

HLA class I and class II associations were examined in relation to measles virus-specific cytokine responses in 339 healthy children who had received two doses of live attenuated measles vaccine. Multivariate linear regression modeling analysis revealed suggestions of associations between the expression of DPA1*0201 (p=0.03) and DPA1*0202 (p=0.09) alleles and interleukin-2 (IL-2) cytokine production (global p-value 0.06). Importantly, cytokine production and DQB1 allele associations (global p-value 0.04) revealed that the alleles with the strongest association with IL-10 secretion were DQB1*0302 (p=0.02), DQB1*0303 (p=0.07) and DQB1*0502 (p=0.06). Measles-specific IL-10 secretion associations approached significance with DRB1 and DQA1 loci (both global p-values 0.08). Specifically, suggestive associations were found between DRB1*0701 (p=0.07), DRB1*1103 (p=0.06), DRB1*1302 (p=0.08), DRB1*1303 (p=0.06), DQA1*0101 (p=0.08), and DQA1*0201 (p=0.04) alleles and measles-induced IL-10 secretion. Further, suggestive association was observed between specific DQA1*0505 (p=0.002) alleles and measles-specific IL-12p40 secretion (global p-value 0.09) indicating that cytokine responses to measles antigens are predominantly influenced by HLA class II genes. We found no associations between any of the alleles of HLA A, B, and Cw loci and cytokine secretion. These novel findings suggest that HLA class II genes may influence the level of cytokine production in the adaptive immune responses to measles vaccine.

Ovsyannikova, Inna G.; Ryan, Jenna E.; Jacobson, Robert M.; Vierkant, Robert A.; Pankratz, V. Shane; Poland, Gregory A.



Biochemical and biologic characterization of lymphocyte regulatory molecules. III. The isolation and phenotypic characterization of Interleukin-2 producing T cell lymphomas.  


To isolate a stable tumor cell line source of Interleukin 2 (IL-2 formerly referred to as T cell growth factor), over 40 murine leukemia and lymphoma cells as well as 9 clonal helper and killer IL-2-driven T cell lines were screened for both constitutive and mitogen-stimulated IL-2 production. A radiation-induced splenic lymphoma from the B10.BR mouse, the LBRM-33 cell line, could be stimulated to produce over 1000 units/ml of IL-2 after 24 hr exposure to T cell mitogens. Peak IL-2 activity was found in supernatants harvested from 24-hr cultures of either 1% PHA or 20 micrograms/ml Con A-stimulated LBRM-33 cells (10(6) cells/ml). IL-2 production observed in both serum-free and serum-containing cultures represented between 1000 and 5000 times the quantity of IL-2 produced in conventional cultures of mitogen-activated rat or mouse spleen cells. Peak IL-2 production by LBRM-33 cultures (stimulated at either optimal Con A or PHA concentrations or co-stimulated with suboptimal amounts of mitogen and phorbol myristate acetate) was consistently accompanied by LBRM-33 cell death. Phenotypic characterization of the producer cell revealed LBRM-33 cells to be Thy 1+, Ly 1+, Ly 2+, Ly 3+, Qa 2-3+, Qa 3.2+, Qat 4+, and Ly 5+. These studies provide further evidence that IL-2 is a T cell product and establish a source of IL-2 that will be a valuable reagent for the isolation and further molecular characterization of this immunoregulatory molecule. PMID:6968790

Gillis, S; Scheid, M; Watson, J



Treatment of HIV-related primary central nervous system lymphoma with azt high dose, HAART, interleukin-2 and foscarnet in three patients  

PubMed Central

Purpose Combined immunomodulatory and antiviral treatment was administered to three patients with newly diagnosed HIV-associated primary central nervous system lymphoma (PCNSL) in an attempt to improve outcomes. Patients and methods Three patients from our institution who were recently diagnosed with HIV-associated PCNSL received intravenous azidothymidine (AZT) 1.6 gr. bid for two weeks, followed by oral AZT 250 mg bid from day 15. In addition, complementary highly active antiretroviral therapy (HAART) with a second nucleoside reverse transcriptase inhibitor (NRTI) plus one protease inhibitor (PI) and interleukin 2 (IL-2) subcutaneously 2 million units twice daily (bid) plus foscarnet 90 mg/kg bid were administered on days 1-14. One patient received anti-EpsteinBarr virus (EBV)-maintenance therapy with ganciclovir, followed by cidofovir [1]. Results All patients experienced progressive disease while on induction therapy, and switched early to whole-brain radiation therapy (WBRT) as second linetreatment. No grade 3 or 4 toxicities were observed. Two patients died on days 50 and 166 respectively due to progressive disease. The third patient with histologically proven lymphoproliferation and only suspected PCNSL remained alive at 53 months. He was on HAART and remained clinically and neurologically stable. Conclusion Although IL-2, HAART, high-dose AZT and foscarnet are used for other HIV-related conditions, they did not demonstrate benefit in lymphoma remission for 2 HIVassociated PCNSL patients. The third patient went into delayed remission after additional radiotherapy and was in good clinical and neurological health status over 53 months after diagnosis.



Selective long-term elimination of natural killer cells in vivo by an anti-interleukin 2 receptor beta chain monoclonal antibody in mice  

PubMed Central

The interleukin 2 receptor beta chain (IL-2R beta) is preferentially expressed in natural killer (NK) cells, but is not detected in a majority of resting T and B cells. We recently established a novel monoclonal antibody (mAb) to murine IL-2R beta and examined in vivo the effect of the mAb in mice. We found that intraperitoneal injection of the anti-IL-2R beta mAb into adult mice resulted in a selective in vivo elimination of splenic NK function in various mouse strains. The reduction of NK cell function is associated with complete disappearance of NK1.1+ cells in C57BL/6 mice. Other lymphocyte subsets in the thymus and spleen were uncompromised. T cell function was not affected by the mAb treatment as judged by allogeneic cytotoxic T cell induction. The single injection of anti-IL-2R beta mAb caused a long-term elimination of splenic NK cells, lasting for at least 5 wk. We also found that NK and/or NK precursor cells become susceptible to the mAb treatment only after birth, suggesting that functional maturation of NK cells in terms of IL-2R beta expression is a later event in the course of NK cell development. The use of the anti-IL-2R beta mAb will be useful in defining the physiological role of NK cells in host defense as well as dissecting their developmental pathway in vivo.



Dehydroepiandrosterone sulfate decreases the interleukin-2-mediated overactivity of the natural killer cell compartment in senile dementia of the Alzheimer type.  


Since dehydroepiandrosterone sulfate (DHEAS) has been involved in the regulation of cellular immunity, the aim of the presence study was to evaluate whether the age-dependent reduction of DHEAS was associated with changes of natural killer (NK) immune function in healthy elderly subjects and in patients with senile dementia of the Alzheimer type (SDAT). Circulating DHEAS was determined throughout 24 h (circadian profile). NK cytotoxic activity was measured as spontaneous and induced cytotoxicity during exposure with DHEAS (10(-7) M), interleukin-2 (IL-2; 100 IU) and IL-2 (100 IU) coincubated with DHEAS (10(-7) M). DHEAS was significantly reduced in healthy elderly subjects (mesor M +/- SD = 2.3 +/- 0.5 micromol/l) and SDAT (1.6 +/- 0.4 micromol/l) patients compared to healthy young subjects (6.7 +/- 0.9 micromol/l; p < 0.001); significant differences were also found when healthy elderly subjects and SDAT patients were compared (p < 0.01). A significant inverse correlation between age and DHEAS levels was demonstrated in SDAT and healthy elderly subjects (p < 0.05). The decrease in 24-hour DHEAS secretion was associated with a higher NK cytotoxic response to DHEAS in the healthy elderly subject group than in healthy subjects of young age (p < 0.01). Increased NK cell activity during IL-2 incubation was found in patients with SDAT in comparison with the healthy elderly subject (p < 0.001). On the contrary, NK cell cytotoxic response of SDAT patients was less pronounced during DHEAS exposure and when DHEAS was coincubated with IL-2 (p < 0.001). These data suggest an immunomodulatory role of DHEAS on NK functional activity in physiological aging and SDAT. The antagonizing effect of DHEAS on NK overactivity during exposure with cytokines might counteract some neuroimmune components related to the pathogenesis and progression of the disease. PMID:9844034

Solerte, S B; Fioravanti, M; Schifino, N; Cuzzoni, G; Fontana, I; Vignati, G; Govoni, S; Ferrari, E


Synergism of interleukin-2 and cyclosporine A in induction of a graft-versus-tumor effect without graft-versus-host disease after syngeneic bone marrow transplantation.  


Interleukin-2 (IL-2) therapy generates killer cells with major histocompatibility complex (MHC)-unrestricted cytotoxicity against most tumors but not normal tissues. Cyclosporine A (CsA) has been reported to break tolerance to self and to induce killer cells with specificity against class II MHC (Ia) antigens both on the host and the tumor cells, resulting in a mild graft-versus-host disease (GVHD) in an autologous bone marrow transplantation (BMT) setting in the rat. We used these two agents in a syngeneic BMT model in a strain of mice that does not develop GVHD with CsA. Therapy with either agent alone was ineffective, whereas a combination of CsA plus IL-2 after BMT induced a potent graft-versus-tumor (GVT) effect against a melanoma and an acute myeloid leukemia. The antitumor effect could be adoptively transferred by infusing spleen cells harvested from mice treated with CsA plus IL-2 into secondary recipients that received chemoradiotherapy. The cytotoxicity of these cells was not influenced by treatment of tumor cells with gamma-interferon or Ia antibody. The cytotoxic effect was mediated by Thy 1+ and asialo GM 1+ cells. There was no GVHD either in the primary recipients of CsA and IL-2 or in those receiving the adoptively transferred spleen cells. Our findings show that combination therapy with CsA and IL-2 after syngeneic BMT induces a potent GVT effect in a non-MHC-restricted manner, and point to the existence of differences between the mechanisms of GVT and GVHD. PMID:1611084

Charak, B S; Agah, R; Mazumder, A



Prodigiosin blocks T cell activation by inhibiting interleukin-2Ralpha expression and delays progression of autoimmune diabetes and collagen-induced arthritis.  


Prodigiosin (PDG) was previously reported to be a T cell-specific immunosuppressant. Here we describe the mechanism of action of PDG in T cells and the effect of PDG on autoimmune diseases. PDG selectively suppresses concanavalin A (Con A)-induced T cell proliferation, but has little effect on lipopolysaccharide-induced proliferation of B cells and nitric oxide production of macrophages. Although PDG does not block interleukin (IL)-2 production, it efficiently inhibits interleukin-2 receptor alpha-chain (IL-2Ralpha) expression, and this results in a disruption of the IL-2/IL-2R signaling pathway, on which a great part of the regulation of T cell activation depends. PDG blocks T cell differentiation into effector helper T cells secreting interferon-gamma and IL-4 as well as into effector cytotoxic T lymphocytes expressing perforin, which is at least in part resulting from inhibition of the IL-2/IL-2R signaling. PDG indirectly blocks signal transducer and activator of transcription activation by inhibiting cytokine signalings in Con A-activated T cells, although it does not inhibit the activation of nuclear factor-kappaB, nuclear factor of activated T cells, and activator protein-1. As direct evidence of immunosuppression in vivo, we show that PDG markedly reduced blood glucose levels and cellular infiltration into the pancreatic islets in nonobese diabetic mice, and that it also delays the onset of collagen-induced arthritis in DBA/1 mice. In conclusion, our results demonstrate that PDG has a unique mode of action, namely, that it blocks T cell activation by inhibiting primarily IL-2Ralpha expression in the IL-2/IL-2R signaling, and show that this compound represents a promising immunosuppressant candidate for the treatment of autoimmune diseases. PMID:11602650

Han, S B; Park, S H; Jeon, Y J; Kim, Y K; Kim, H M; Yang, K H



Phase I\\/II open-label study of the biologic effects of the interleukin-2 immunocytokine EMD 273063 (hu14.18IL2) in patients with metastatic malignant melanoma  

Microsoft Academic Search

BACKGROUND: To explore the biological activity of EMD 273063 (hu14.18-IL2), a humanized anti-GD2 monoclonal antibody fused to interleukin-2 (IL2), in patients with unresectable, stage IV cutaneous melanoma as measured by induction of immune activation at the tumor site and in peripheral blood. METHODS: Nine patients were treated with 4 mg\\/m2 per day of EMD 273063 given as a 4-h intravenous

Antoni Ribas; John M Kirkwood; Michael B Atkins; Theresa L Whiteside; William Gooding; Andreas Kovar; Stephen D Gillies; Oscar Kashala; Michael A Morse



Activation of Jak\\/STAT Proteins Involved in Signal Transduction Pathway Mediated by Receptor for Interleukin 2 in Malignant T Lymphocytes Derived from Cutaneous Anaplastic Large T-Cell Lymphoma and Sezary Syndrome  

Microsoft Academic Search

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak\\/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and

Qian Zhang; Irena Nowak; Eric C. Vonderheid; Alain H. Rook; Marshall E. Kadin; Peter C. Nowell; Leslie M. Shaw; Mariusz A. Wasik



Serum levels of soluble Fas, soluble tumor necrosis factor-receptor II, interleukin-2 receptor and interleukin-8 as early predictors of hepatocellular carcinoma in Egyptian patients with hepatitis C virus genotype-4  

Microsoft Academic Search

BACKGROUND: Liver disease progression from chronic hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) is associated with an imbalance between T-helper 1 and T-helper 2 cytokines. Evaluation of cytokines as possible candidate biomarkers for prediction of HCC was performed using soluble Fas (sFas), soluble tumor necrosis factor receptor-II (sTNFR-II), interleukin-2 receptor (IL-2R) and interleukin-8 (IL-8). RESULTS: The following patients

Abdel-Rahman N Zekri; Hanaa M Alam El-Din; Abeer A Bahnassy; Naglaa A Zayed; Waleed S Mohamed; Suzan H El-Masry; Sayed K Gouda; Gamal Esmat



Hierarchy of Protein Tyrosine Kinases in Interleukin2 (IL2) Signaling: Activation of Syk Depends on Jak3; However, Neither Syk nor Lck Is Required for IL2-Mediated STAT Activation  

Microsoft Academic Search

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation,




Interleukin 2 is not sufficient as helper component for the activation of cytotoxic T lymphocytes but synergizes with a late helper effect that is provided by irradiated T-region-incompatible stimulator cells  

SciTech Connect

Interleukin 2-containing supernatants from concanavalin A-activated spleen cells (CSCS) were found to provide strong helper activity for cytotoxic T lymphocyte (CTL) responses against allogeneic stimulator cells in microculture systems, but provided usually insufficient help for CTL responses against l-region compatible allogeneic or TNP-haptenated syngeneic stimulator cells. The interleukin 2-containing supernatant from HGG-activated AODH 7.1 hybridoma cells also mediated only relatively weak CTL responses against TNP-haptenated syngeneic cells in microcultures. Both types of supernatants, however, supported substantial responses against TNP-haptenated syngeneic stimulator cells if irradiated allogeneically activated syngeneic T cells or irradiated allogeneic spleen cells were added to the cultures. The allogeneic cells and the activated syngeneic T cells provided little helper activity if they were added in the absence of the interleukin 2-containing supernatants, thus demonstrating a synergistic effect between these 2 helper components. An l-region difference was sufficient for the helper effect of the allogeneic cells and control experiments showed that the presence of foreign l-region determinants could not be substituted for the TNP-haptenated stimulator cells.

Reddehase, M.; Suessmith, W.; Moyers, C.; Falk, W.; Droege, W.



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

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.

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



Pseudo-high affinity interleukin 2 (IL-2) receptor lacks the third component that is essential for functional IL-2 binding and signaling  

PubMed Central

Functional studies of the interleukin 2 receptor (IL-2R) of two (ED515- D and Kit225) IL-2-dependent and three (ED515-I, 3T3-alpha beta 11, and Hut102) IL-2-independent cell lines were done. All of these cell lines appeared to express high as well as low affinity IL-2R. However, ED515- I and 3T3-alpha beta 11, which expressed the IL-2R beta chain, did not bind IL-2 at all when IL-2 binding to their IL-2R alpha chain was blocked with anti-Tac monoclonal antibody, whereas the intermediate affinity binding in ED515-D, Kit225, and Hut102 cells remained. We tentatively called the high affinity IL-2R of the former cells pseudo- high affinity IL-2R. The dissociation constant of pseudo-high affinity IL-2R was higher than that of ordinary high affinity IL-2R. Internalization of cell-bound 125I-IL-2 into ED515-I and 3T3-alpha beta 11 cells was less efficient than that into ED515-D cells. The addition of IL-2 neither promoted cell growth nor upregulated IL-2R alpha chain expression in ED515-I and 3T3-alpha beta 11 cells. Furthermore, tyrosine phosphorylation of the cellular proteins (p120, p98, p96, p54, and p38) was induced or enhanced in response to the addition of IL-2 in ED515-D and Kit225 cells, but not in the cell lines expressing pseudo- high affinity IL-2R. Finally, 125I-IL-2 crosslinking followed by SDS- PAGE analysis showed an 80-kD band corresponding to p65 + IL-2, in addition to bands corresponding to IL-2R alpha and beta chain + IL-2 in cells bearing ordinary high affinity IL-2R but not in cells with pseudo- high affinity IL-2R. Taken together, we consider that another protein whose molecular mass is approximately 65 kD is functionally important in IL-2 binding and subsequent signal transduction and may be the third component of IL-2R.



Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein  

PubMed Central

Background Fluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis. Results Hybrid genes were constructed, which encode either the mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG) fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin) and the respective sera were analysed using MOG-displaying PHA granules and FACS analysis showing a specific and sensitive detection of antigen-specific antibodies within a wide dynamic range. Conclusion E. coli can be genetically engineered to produce PHA granules displaying correctly folded eukaryotic proteins and which can be applied as beads in FACS based diagnostics. Since PHA granule formation and protein attachment occurs in one step already inside the bacterial cell, microbial production could be a cheap and efficient alternative to commercial beads.

Backstrom, B Thomas; Brockelbank, Jane A; Rehm, Bernd HA



Immunopharmacology and cytokine production of a low-dose schedule of intraperitoneally administered human recombinant interleukin-2 in patients with advanced epithelial ovarian carcinoma.  


We determined in the peritoneal cavity (p.c.) of epithelial ovarian carcinoma patients during a 4-day treatment cycle of low-dose recombinant human interleukin-2 (rIL-2): (a) pharmacokinetics of IL-2, (b) endogenous cytokine production, and (c) numbers and percentages of peritoneal exudate lymphocytes. We administered 6 x 10(5) IU/m2 of rIL-2 (0.03 mg/m2 Proleukin rIL-2) intraperitoneally (i.p.) over 30 min on each of 4 days. One and one-half liters of D5 0.25 NS was injected i.p. before each rIL-2 infusion. Multiple peritoneal fluid samples were obtained from each of four patients on day 1 and day 4 for detection of IL-2, endogenous cytokines, and soluble IL-2 receptor (IL-2R-alpha). IL-2 concentrations in the peritoneal fluid were determined by bioassay and interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-10, transforming growth factor (TGF)-beta 2, and sIL-2R-alpha by enzyme-linked immunosorbent assay. Numbers of cells per microliter and lymphocyte subpopulation percentages after staining with a panel of monoclonal antibodies were determined on day 1, day 4, and subsequent off-treatment days. IL-2 disappearance in the p.c. was well described by a pharmacokinetic model having constant-rate infusion and biexponential disposition. About 90% of the IL-2 disappearance occurred during the beta-phase, during which IL-2 concentrations were sustained at approximately 10-30 ng/ml (day 1 and day 4) and the median t1/2 beta was 21.5 and 9.2 h on days 1 and 4, respectively. In four of four patients, p.c. production of IL-10 was observed on day 1 and day 4 (maximum 387 pg/ml). Maximum levels of IFN-gamma and sIL-2R-alpha were observed on day 4. (IFN-gamma 217 pg/ml; sIL2-R-alpha: 3486 U/ml). No increases in TNF-alpha or TGF-beta 2 were observed. Large increases in p.c. CD3+, CD4+, CD8+, CD16+, and CD56+ cells were observed. We conclude that biologically active levels of IL-2 are generated in p.c. fluids after i.p. administration of rIL-2 at 0.03 mg/m2. PMID:9041464

Freedman, R S; Gibbons, J A; Giedlin, M; Kudelka, A P; Kavanagh, J J; Edwards, C L; Carrasco, C H; Nash, M A; Platsoucas, C D



Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1.  


Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells. PMID:11435608

Biola, A; Lefebvre, P; Perrin-Wolff, M; Sturm, M; Bertoglio, J; Pallardy, M



Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and lymphoma.  


Natural killer-like T lymphocytes termed cytokine-induced killer (CIK) cells have been shown to eradicate established tumours in a severe combined immune deficient (SCID) mouse/human lymphoma model. Recently, we demonstrated that CIK cells transfected with cytokine genes possess an improved proliferation rate and a significantly higher cytotoxic activity as compared to non-transfected cells. Here, in a phase I clinical protocol, autologous CIK cells were generated from peripheral blood obtained by leukapheresis in patients with metastatic renal cell carcinoma, colorectal carcinoma and lymphoma. CIK cells were transfected with a plasmid containing the interleukin-2 (IL-2) gene via electroporation. Transfected cells generated IL-2 in the range of 330-1800 pg 10(-6) cells 24 h(-1) with a mean of 836 pg 10(-6) cells 24 h(-1). Ten patients received 1-5 intravenous infusions of IL-2-transfected CIK cells; five infusions with transfected CIK cells were given. In addition, the same patients received five infusions with untransfected CIK cells for control reasons. In three patients, WHO grade 2 fever was observed. Based on polymerase chain reaction of peripheral blood transfected cells could be detected for up to 2 weeks after infusion. There was a significant increase in serum levels of interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta) during treatment. Interestingly, there was also an increase in CD3+ lymphocytes in the blood of patients during therapy. In accordance, a partial increase in cytotoxic activity in peripheral blood lymphocytes (PBLs) was documented when patient samples before and after therapy were compared. Concerning clinical outcome, six patients remained in progressive disease, three patients showed no change by treatment, and one patient with lymphoma developed a complete response. In conclusion, we were able to demonstrate that CIK cells transfected with the IL-2 gene can be administered without major side-effects and are promising for future therapeutic trials. PMID:10576658

Schmidt-Wolf, I G; Finke, S; Trojaneck, B; Denkena, A; Lefterova, P; Schwella, N; Heuft, H G; Prange, G; Korte, M; Takeya, M; Dorbic, T; Neubauer, A; Wittig, B; Huhn, D



A phase I study of recombinant human interleukin-2 and alpha-interferon-2a in patients with renal cell cancer, colorectal cancer, and malignant melanoma.  


Preclinical data suggest synergy of interleukin-2 (IL-2) combined with alpha-interferon (IFN). In addition, toxicities of IL-2 may be decreased by intermittent continuous infusion. The purpose of this trial was to determine the maximum tolerated dose (MTD) of recombinant IL-2 combined with alpha-IFN in patients with renal cancer, colon cancer, melanoma, and malignant B-cell disease. IL-2 was given by continuous i.v. infusion at an initial dose of 5 X 10(5) units (U)/m2/d for 4 days plus IFN at 6 X 10(6) U/m2/d intramuscularly days 1 and 4 weekly for 4 weeks. Patients who achieved a response or stable disease received an additional 4 weeks of therapy. IL-2 doses were increased to 1, 2, 3, 5, and 7 X 10(6) U/m2/d with three to eight patients at each dose level, at each of the two participating institutions. The dose of IFN was 6 X 10(6) U/m2 days 1 and 4 for all but five patients whose IFN dose was doubled to 12 X 10(6) U/m2/d. Forty-three patients were entered on this study with 34 completing at least 4 weeks of therapy. Six patients were taken off study because of Grades III or IV pulmonary, neurologic, or cardiac toxicity; one for progressive disease; one for CNS metastases, and one for personal reasons. All of the toxicities were reversible. Chills and fever were universal, especially on days 1 and 4. Mild and moderate nausea, vomiting, diarrhea, anorexia, malaise, and cutaneous erythema were present in most patients. Fluid retention and occasional pleural effusions were observed at the higher IL-2 doses but were not dose-limiting. Significant hypotension associated with oliguria was seen, and these patients were treated with vasopressors and colloids. None of the patients required ICU admission. Thirty-four patients were evaluable for response. There were 4/18 (22%) renal cell patients who experienced a partial response. No responses were seen in patients with melanoma, lymphoma, or colorectal cancer. The combined debilitating symptoms of fatigue, diarrhea, hypotension, fluid retention, and anorexia defined the MTD as 5 X 10(6) U/m2/d of IL-2 and 6 X 10(6) U/m2 of alpha-IFN. PMID:2386896

Mittelman, A; Huberman, M; Puccio, C; Fallon, B; Tessitore, J; Savona, S; Eyre, R; Gafney, E; Wick, M; Skelos, A



Increased excretion of soluble interleukin 2 receptors and free light chain immunoglobulins in the urine of patients with active lupus nephritis.  

PubMed Central

Samples of protein from the urine of 23 patients with lupus nephropathy and 15 patients with proteinuria who did not have systemic lupus erythematosus (SLE) were studied for the presence of cytokines, soluble interleukin 2 receptors (sIL-2R), and free light chain immunoglobulins. The patients with lupus nephropathy were divided into two groups with active (nephritis) and inactive inflammation (nephrosis) based on the results of the analysis of urine samples and renal histology. The crude urine proteins (5 mg/ml) after precipitation by 80% ammonium sulphate from 14 patients with lupus nephritis contained higher concentrations of sIL-2R (4.88 (SEM 1.27 ng/ml) than those from nine patients with nephrosis (1.11 (0.52) ng/ml) or 15 patients without SLE (1.31 (0.87) ng/ml). The concentration of sIL-2R in protein from urine samples was not correlated with the concentration in plasma and was inversely correlated with the excretion of protein in urine over 24 hours in patients with SLE. It is suggested that, in addition to leakage from the circulation, the local production of sIL-2R by inflamed kidneys is possible. The crude proteins in urine were further fractionated by gel filtration on Sephacryl S-200. Arbitrarily, four fractions could be obtained from urine from patients with SLE but only three fractions were found in the urine of patients without SLE. Fraction IV derived from patients with nephritis or nephrosis augmented the pokeweed mitogen induced [3H]thymidine uptake of mononuclear cells. In addition, the positive rates of free kappa (kappa) (35.7%) and lambda (lambda) (42.9%) chains in proteins in urine from nephritic patients were higher than those in the other two groups. These results suggest that the severity of inflammation in the kidneys of patients with lupus can be reflected by the increased excretion of sIL-2R, free light chain immunoglobulins, and cytokine-like molecules in urine. Images

Tsai, C Y; Wu, T H; Sun, K H; Lin, W M; Yu, C L



Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and lymphoma  

PubMed Central

Natural killer-like T lymphocytes termed cytokine-induced killer (CIK) cells have been shown to eradicate established tumours in a severe combined immune deficient (SCID) mouse/human lymphoma model. Recently, we demonstrated that CIK cells transfected with cytokine genes possess an improved proliferation rate and a significantly higher cytotoxic activity as compared to non-transfected cells. Here, in a phase I clinical protocol, autologous CIK cells were generated from peripheral blood obtained by leukapheresis in patients with metastatic renal cell carcinoma, colorectal carcinoma and lymphoma. CIK cells were transfected with a plasmid containing the interleukin-2 (IL-2) gene via electroporation. Transfected cells generated IL-2 in the range of 330–1800 pg 10?6 cells 24 h?1 with a mean of 836 pg 10?6 cells 24 h?1. Ten patients received 1–5 intravenous infusions of IL-2-transfected CIK cells; five infusions with transfected CIK cells were given. In addition, the same patients received five infusions with untransfected CIK cells for control reasons. In three patients, WHO grade 2 fever was observed. Based on polymerase chain reaction of peripheral blood transfected cells could be detected for up to 2 weeks after infusion. There was a significant increase in serum levels of interferon gamma (IFN-?), granulocyte–macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-?) during treatment. Interestingly, there was also an increase in CD3+ lymphocytes in the blood of patients during therapy. In accordance, a partial increase in cytotoxic activity in peripheral blood lymphocytes (PBLs) was documented when patient samples before and after therapy were compared. Concerning clinical outcome, six patients remained in progressive disease, three patients showed no change by treatment, and one patient with lymphoma developed a complete response. In conclusion, we were able to demonstrate that CIK cells transfected with the IL-2 gene can be administered without major side-effects and are promising for future therapeutic trials. © 1999 Cancer Research Campaign

Schmidt-Wolf, I G H; Finke, S; Trojaneck, B; Denkena, A; Lefterova, P; Schwella, N; Heuft, H-G; Prange, G; Korte, M; Takeya, M; Dorbic, T; Neubauer, A; Wittig, B; Huhn, D



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


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 outpatient chemo-/immunotherapy is feasible and results in objective response rates and survival similar to earlier trials. Pretreatment risk, as defined by serum lactate dehydrogenase (LDH) and performance status, has a significant impact on treatment outcome and patient survival. PMID:9792153

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



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

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 outpatient chemo-/immunotherapy is feasible and results in objective response rates and survival similar to earlier trials. Pretreatment risk, as defined by serum lactate dehydrogenase (LDH) and performance status, has a significant impact on treatment outcome and patient survival.

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



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

PubMed Central

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

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



Widespread inosine-containing mRNA in lymphocytes regulated by ADAR1 in response to inflammation  

PubMed Central

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of pre-mRNA catalysed by an RNA-specific adenosine deaminase (ADAR). A-to-I RNA editing has been previously reported in the pre-mRNAs of brain glutamate and serotonin receptors and in lung tissue during inflammation. Here we report that systemic inflammation markedly induces inosine-containing mRNA to approximately 5% of adenosine in total mRNA. Induction was the result of up-regulation of A-to-I RNA editing as both dsRNA editing activity and ADAR1 expression were increased in the spleen, thymus and peripheral lymphocytes from endotoxin-treated mice. Up-regulation of ADAR1 was confirmed in vitro in T lymphocytes and macrophages stimulated with a variety of inflammatory mediators including tumour necrosis factor-? and interferon-?. A late induction of RNA editing was detected in concanavalin A-activated splenocytes stimulated with interleukin-2 in vitro. Taken together, these data suggest that a large number of inosine-containing mRNAs are produced during acute inflammation via up-regulation of ADAR1-mediated RNA editing. These events may affect the inflammatory and immune response through modulation of protein production.

Yang, Jing-Hua; Luo, Xiaoxing; Nie, Yongzhan; Su, Yingjun; Zhao, Qingchuan; Kabir, Koroush; Zhang, Dexin; Rabinovici, Reuven



Cytokine mRNA expression profiles in rats infected with the intestinal nematode Nippostrongylus brasiliensis.  

PubMed Central

Although the immune responses to intestinal nematode infection have been well studied and have been shown to be strongly driven by Th2-associated cytokines in mice, such information has been limited with respect to rats. We investigated changes in levels of the mRNAs encoding interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-10, and gamma interferon in the mesenteric lymph nodes of rats infected with Nippostrongylus brasiliensis by reverse transcription-PCR in comparison with immunoglobulin E (IgE)/IgG2a antibody, eosinophil, basophil, and mucosal mast cell responses. In the two rat strains used, Brown Norway and Fischer-344, which show different responses to allergens, serum IgE increased to much higher levels in the former than in the latter 2 weeks after infection. Intestinal mastocytosis was observed much earlier and more intensely in Brown Norway rats than in Fischer-344 rats, but the degrees of peripheral eosinophilia and basophilia did not differ between the two strains. In both strains, IL-3, IL-4, and IL-5 mRNA expression increased and peaked around 7 to 14 days after infection, while expression of IL-2, IL-10, and gamma interferon mRNAs did not change notably throughout the experimental period. The highest IL-4 mRNA expression was observed slightly earlier in Brown Norway than in Fischer-344 rats, but levels of IL-3 and IL-5 mRNAs peaked synchronously in both strains. The amounts of mRNAs encoding these three cytokines were always higher in Brown Norway than in Fischer-344 rats. It is suggested that in rats, Th2 or Th2-like cells are also induced after nematode infection, and IgE elevation is mainly related to increased IL-4 gene expression.

Matsuda, S; Uchikawa, R; Yamada, M; Arizono, N



The combination of interleukin-2 and interferon effectively augments the antibody-dependent cellular cytotoxicity of monoclonal antibodies 17-1A and BR55-2 against the colorectal carcinoma cell line HT29  

Microsoft Academic Search

Monoclonal antibodies (mAb) are promising substances for the treatment of colorectal carcinoma, but the efficiency of this\\u000a therapy still needs further improvement. We used a flow-cytometric cytotoxicity test to determine the efficacy of the cytokines\\u000a interferon ? (IFN?) and ? (IFN?), interleukin-2 (IL-2), macrophage-colony-stimulating factor (M-CSF), granulocyte\\/macrophage-CSF\\u000a (GM-CSF) and tumor necrosis factor ? (TNF?) in enhancing the antibody-dependent cellular cytoxicity

Sven Bungard; Dimitri Flieger; Susann Schweitzer; Tilman Sauerbruch; Ulrich Spengler



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

Microsoft Academic Search

We carried out an open, randomized, phase III, multicenter clinical trial to compare, in neo-adjuvant setting, the clinical\\u000a response and toxicity of the combination chemotherapy cisplatin?+?5-FU with the same combination plus s.c. recombinant interleukin-2\\u000a (rIL-2) in patients with advanced (stage III–IV) head and neck squamous-cell carcinoma (HNSCC). Regimen A was the classical\\u000a Al Sarraf treatment: 100?mg\\/m2 cisplatin i.v. on day

Giovanni Mantovani; Vittorio Gebbia; Mario Airoldi; Cesare Bumma; Paolo Contu; Alessandro Bianchi; Massimo Ghiani; Daniela Dessì; Elena Massa; Luigi Curreli; Biancarosa Lampis; Paola Lai; Carlo Mulas; Antonio Testa; Ernesto Proto; Gabrio Cadeddu; Giorgio Tore



Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization.  


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

Zenklusen, Daniel; Singer, Robert H



Degradation of mRNA in eukaryotes  

Microsoft Academic Search

Based on the above mechanisms of mRNA degradation, an integrated model of mRNA turnover can be proposed (Figure 1). In this model, all polyadenylated mRNAs would be degraded by the deadenylation-dependent pathway at some rate. In addition to this default pathway, another layer of complexity would come from degradation mechanisms specific to individual mRNAs or to classes of mRNAs. Such

Clare A Beelman; Roy Parker



Depolarization regulates adrenal preproenkephalin mRNA.  

PubMed Central

The regulation of neuropeptide gene expression has been investigated by using rat adrenal medullae grown in explant culture. After 3 days in culture the (now denervated) explants exhibited a 10-fold increase in leucine-enkephalin (leu-EK) content. Inhibition of protein synthesis with cycloheximide completely blocked the rise, whereas inhibition of RNA synthesis with actinomycin D or alpha-amanitin inhibited the increase by 50%. Inhibition of DNA synthesis with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside) had no discernible effect. To determine whether the rise in leu-EK was associated with an increase in specific mRNA coding for the opiate peptide precursor, blot hybridization analysis was performed. A single species of preproenkephalin mRNA was detected after various culture periods. The amount of mRNA increased 34-fold after 2 days in culture and 74-fold after 4 days. Consequently, the rise in mRNA levels preceded the increase in the amount of leu-EK. Depolarization of the adrenal medullae with either elevated potassium or veratridine, which prevents leu-EK accumulation, inhibited the increase in the amount of preproenkephalin mRNA. Moreover, the effect of veratridine was blocked by tetrodotoxin, suggesting that transmembrane sodium ion influx affects the increase in the amount of message. Our studies suggest that elevation of leu-EK in explanted (denervated) medullae is associated with increased amounts of mRNA coding for the peptide precursor and that these processes can be regulated by depolarization. Images

LaGamma, E F; White, J D; Adler, J E; Krause, J E; McKelvy, J F; Black, I B



mRNA export: threading the needle.  


After mRNA biogenesis, several proteins interact with the messenger to ensure its proper export to the cytoplasm. Some of these proteins will bind RNA early on, at the onset of transcription by RNA polymerase II holoenzyme, while others will join later for downstream processing steps, such as poly-adenylation or splicing, or may direct mRNA ribonucleoprotein particle migration to the nucleopore. We recently discovered that Arabidopsis plant knockout for the protein MOS11 (MODIFIER OF SNC1, 11) partially suppresses autoimmune responses observed in the TNL-type [TIR/NBS/LRR (Toll-interleukin-like receptor/nucleotide-binding site/C-terminal leucine-rich repeat)] R gene gain-of-function variant snc1 (suppressor of npr1-1, constitutive 1). This suppression of resistance to pathogens appears to be caused by a decrease in nuclear mRNA export in mos11-1 snc1 plants. In humans, the putative ortholog of MOS11, CIP29 (29-kDa cytokine-induced protein), interacts with three proteins that are also involved in mRNA export: DDX39 (DEAD-box RNA helicase), TAF15 of the FUS family (FUSED IN SARCOMA), and ALY (ALWAYS EARLY), a protein implicated in mRNA export in mammalian systems. These proteins have received very little attention in plants. Here, we will discuss their particularities and role in mRNA export and biotic stress. PMID:23526740

Gaouar, Ouassila; Germain, Hugo



Elevated intracellular calcium increases ferritin H expression through an NFAT-independent post-transcriptional mechanism involving mRNA stabilization.  


An increase in intracellular Ca2+ is one of the initiating events in T-cell activation. A calcium-mediated signalling cascade in T-cells involves activation of calcineurin and the dephosphorylation and translocation of NFAT (nuclear factor of activated T-cells), resulting in the transcriptional activation of target genes such as IL-2 (interleukin-2). In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an ARE (antioxidant-responsive element). The facts that the ferritin H ARE contains a composite AP-1 (activator protein 1) site and that NFAT collaborates with AP-1 transcription factors led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T-cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Although NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter after ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT-binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability after actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through post-transcriptional stabilization of the ferritin H mRNA. PMID:18076382

MacKenzie, Elizabeth L; Tsuji, Yoshiaki



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

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.

Kaplan, Barbara L.F. [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Ouyang Yanli [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Herring, Amy [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Yea, Sung Su [Department of Biochemistry, College of Medicine, Inje University, Pusan 614-735 (Korea, Republic of); Razdan, Raj [Organix Inc., Woburn, MA 01801 (United States); Kaminski, Norbert E. [Department of Pharmacology and Toxicology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States)]. E-mail:



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

PubMed Central

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

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



Tumor necrosis factor. alpha. induces proteins that bind specifically to. kappa. B-like enhancer elements and regulate interleukin 2 receptor. alpha. -chain gene expression in primary human T lymphocytes  

SciTech Connect

The authors have investigated the biochemical basis for the activation of interleukin 2 receptor {alpha}-subunit (IL-2R{alpha}) gene expression in primary human T lymphocytes by a cytokine (tumor necrosis factor {alpha}), a T-cell mitogen (phorbol 12-myristate 13-acetate), and the transactivator protein (Tax) from the type I human T-cell leukemia virus. Using in vivo transfection techniques specifically designed for these primary T cells in conjunction with in vitro gel retardation and DNA footprinting assays, the authors found that activation of the IL-2R{alpha} promoter by each of these agents involves the induction of nuclear proteins that specifically interact with a {kappa}B-like enhancer element. DNA-protein crosslinking studies revealed that primary T cells express at least three different inducible DNA-binding proteins that specifically interact with this IL-2R{alpha} {kappa}B element.

Lowenthal, J.W.; Ballard, D.W.; Boehnlein, E.; Greene, W.C. (Duke Univ. Medical Center, Durham, NC (USA))



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

PubMed Central

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.

Singh, Ajita V.; Bandi, Madhavi; Raje, Noopur; Richardson, Paul; Palladino, Michael A.



JNK-mediated Phosphorylation of Cdc25C Regulates Cell Cycle Entry and G2/M DNA Damage Checkpoint*  

PubMed Central

c-Jun NH2-terminal Kinases (JNKs) play a central role in the cellular response to a wide variety of stress signals. After their activation, JNKs induce phosphorylation of substrates, which control proliferation, migration, survival, and differentiation. Recent studies suggest that JNKs may also play a role in cell cycle control, although the underlying mechanisms are largely unexplored. Here we show that JNK directly phosphorylates Cdc25C at serine 168 during G2 phase of the cell cycle. Cdc25C phosphorylation by JNK negatively regulates its phosphatase activity and thereby Cdk1 activation, enabling a timely control of mitosis onset. Unrestrained phosphorylation by JNK, as obtained by a cell cycle-stabilized form of JNK or as seen in some human tumors, results in aberrant cell cycle progression. Additionally, UV irradiation-induced G2/M checkpoint requires inactivation of Cdc25C by JNK phosphorylation. JNK phosphorylation of Cdc25C as well as Cdc25A establishes a novel link between stress signaling and unperturbed cell cycle and checkpoint pathways.

Gutierrez, Gustavo J.; Tsuji, Toshiya; Cross, Janet V.; Davis, Roger J.; Templeton, Dennis J.; Jiang, Wei; Ronai, Ze'ev A.



Evodiamine, a plant alkaloid, induces calcium/JNK-mediated autophagy and calcium/mitochondria-mediated apoptosis in human glioblastoma cells.  


Glioblastomas, the most common primary gliomas, are characterized by increased invasion and difficult therapy. Major clinical medicines for treating gliomas merely extend the survival time for a number of months. Therefore, development of new agents against gliomas is important. Autophagy, a process for degrading damaged organelles and proteins, is an adaptive response to environmental stress. However, the role of autophagy in glioblastoma development still needs to be further investigated. Evodiamine, a major alkaloid isolated from Evodia rutaecarpa Bentham, has various pharmacological activities, such as inhibiting tumor growth and metastatic properties. However, the effects of evodiamine on glioblastomas and their detailed molecular mechanisms and autophagy formation are not well understood. In this study, we observed that evodiamine induced dose- and time-dependent apoptosis in glioma cells. Blockade of calcium channels in endoplasmic reticulum (ER) significantly reduced evodiamine-induced cytosolic calcium elevation, apoptosis, and mitochondrial depolarization, which suggests that evodiamine induces a calcium-mediated intrinsic apoptosis pathway. Interestingly, autophagy was also enhanced by evodiamine, and had reached a plateau by 24h. Pharmacological inhibition of autophagy resulted in increased apoptosis and reduced cell viability. Inhibition of ER calcium channel activation also significantly reduced evodiamine-induced autophagy. Inactivation of c-Jun N-terminal kinases (JNK) suppressed evodiamine-mediated autophagy accompanied by increased apoptosis. Furthermore, evodiamine-mediated JNK activation was abolished by BAPTA-AM, an intracellular calcium scavenger, suggesting that evodiamine mediates autophagy via a calcium-JNK signaling pathway. Collectively, these results suggest that evodiamine induces intracellular calcium/JNK signaling-mediated autophagy and calcium/mitochondria-mediated apoptosis in glioma cells. PMID:23774672

Liu, Ann-Jeng; Wang, Sheng-Hao; Chen, Ku-Chung; Kuei, Hsiu-Ping; Shih, Yung-Luen; Hou, Sz-Ying; Chiu, Wen-Ta; Hsiao, Sheng-Huang; Shih, Chwen-Ming



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)

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.

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



Targeting of locus ceruleus noradrenergic neurons expressing human interleukin-2 receptor ?-subunit in transgenic mice by a recombinant immunotoxin anti-Tac(Fv)-PE38: a study for exploring noradrenergic influence upon anxiety-like and depression-like behaviors.  


The noradrenergic (NA) neurons in the locus ceruleus (LC) were ablated with a high degree of selectivity by immunotoxin-mediated neuronal targeting. Transgenic mice were used in which the human interleukin-2 receptor-? subunit (hIL-2R?; Tac) is expressed under the promoter of dopamine ?-hydroxylase. The recombinant immunotoxin, which is composed of the Fv fragment of an anti-hIL-2R? monoclonal antibody fused to a truncated form of Pseudomonas exotoxin [anti-Tac(Fv)-PE38], was injected bilaterally into the LC of the mouse. As a result, the LC-NA neurons disappeared almost completely, and tissue noradrenaline was depleted in brain regions that receive NA inputs from the LC. The decrement of tissue noradrenaline content was more profound compared with that in mice treated with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), a neurotoxin capable of ablating axons originating from the LC-NA neurons. Mice treated with either the immunotoxin or DSP-4 presented increased anxiety-like behaviors; in contrast, only the immunotoxin-treated mice, and not the DSP-4-treated mice, showed increased depression-like behavior. The immunotoxin-mediated neuronal targeting may provide a means for further unraveling the links between the LC and pathological manifestations of neurological disorders. PMID:21508238

Itoi, Keiichi; Sugimoto, Naoya; Suzuki, Saya; Sawada, Keisuke; Das, Gopal; Uchida, Katsuya; Fuse, Toshimitsu; Ohara, Shinji; Kobayashi, Kazuto



Differential regulation of the protein tyrosine kinase activity following interleukin-2 (IL-2), interferron gamma (IFN-gamma) and SRBC administration in brain tumor-induced conditions: SRBC acting as a dual potentiator in regulating the cytokine profile.  


Protein tyrosine kinases (PTKs) act as an important class of signal transducer in cytokine mediated signaling. Defects in phosphorylation of tyrosine residues of intracellular substrates of the immunocytes are a noted phenomenon in glioma induced immune suppression. Administration of BRMs like Interleukin2 (IL-2), Interferon gamma (IFN-gamma) and SRBC in glioma induced experimental models, improved their survival status by immune potentiation. It was shown that SRBC exerts the maximum anti-tumor immune boosting by augmenting the functional status of the two immunocytes-microglia and lymphocytes when compared with IL-2 and IFN-gamma. The present study focuses on the differential modulation of the protein tyrosine kinase activity in lymphocytes and microglia following the administration of the 3 BRMs. Our findings indicate that PTKs actively transduce signals on administration of exogenous IL-2. But exogenous IFN-gamma administration fails to elicit the enzyme activity. With SRBC administration, a differential PTK activity modulation was observed in the two immunocytes. SRBC not only shifted the cytokine profile to Th1 subset of lymphocytes but also simultaneously upregulated the expression of the activation marker IL-2Ralpha/CD25 thereby resulting in auto-activation of the hosts immunocytes. PMID:15197346

Bhattacharjee, M; Sarkar, S; Dutta, S; Begum, Z; Roy, U R; Chaudhuri, Samares; Chaudhuri, Swapna



Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.  

PubMed Central

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Kawahara, A; Minami, Y; Miyazaki, T; Ihle, J N; Taniguchi, T



Functional coupling of the src-family protein tyrosine kinases p59fyn and p53/56lyn with the interleukin 2 receptor: implications for redundancy and pleiotropism in cytokine signal transduction.  

PubMed Central

The binding of interleukin 2 (IL-2) to the IL-2 receptor (IL-2R) induces a rapid increase in tyrosine phosphorylation of cellular proteins. In a previous study, we have shown that p56lck (lck), a src-family protein tyrosine kinase (src-PTK), physically and functionally associates with the IL-2R beta chain (IL-2R beta). To further investigate a role of src-PTKs in IL-2 signaling, we analyzed a mouse pro-B-cell line, in which lck is not expressed detectably. We observed that in this cell line, IL-2 induces activation of at least two src-PTKs, p59fyn (fyn) and p53/56lyn (lyn). Interestingly, stimulation of this cell line with IL-3 also induces activation of src-PTKs. The activation of fyn or lyn seems to be selective for stimulation with IL-2 or IL-3 since stimulation with IL-6 fails to activate them. Furthermore, we provide evidence for the physical association of fyn with IL-2R beta. Taken together with previous results, our current study suggests that different src-PTKs, each of which is expressed in a cell-type-specific manner, can participate in the IL-2 signal transduction. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

Kobayashi, N; Kono, T; Hatakeyama, M; Minami, Y; Miyazaki, T; Perlmutter, R M; Taniguchi, T



Mouse interleukin-2 structure-function studies: substitutions in the first alpha-helix can specifically inactivate p70 receptor binding and mutations in the fifth alpha-helix can specifically inactivate p55 receptor binding.  

PubMed Central

The function of two alpha-helical regions of mouse interleukin-2 were analyzed by saturation substitution analysis. The functional parts of the first alpha-helix (A) was defined as residues 31-39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha-helix A, Leu31, Asp34, Leu35 and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha-helix A. Residues 31 and 35 had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha-helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha-helix E region demonstrated that alpha-helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha-helix E was important only for the single intramolecular disulfide linkage.

Zurawski, S M; Zurawski, G



Cell fate conversion by mRNA.  


Recent development of a synthetic mRNA-based technology for efficient reprogramming to pluripotency and cell fate conversion without any modification to the genome has generated great interest among researchers and clinicians alike. It is hoped that this technology could contribute to unmet needs on several fronts of regenerative medicine, including mechanistic study of reprogramming, generation of safe induced pluripotent stem cells suitable for clinical applications, and derivation of desired cell types for cell-replacement therapy. We will discuss the technological advancements made by this synthetic mRNA methodology, its implications, as well as the challenges that lie ahead in the field of regenerative medicine. PMID:21345255

Li, Mo; Sancho-Martinez, Ignacio; Izpisua Belmonte, Juan Carlos



Exon repetition in mRNA  

PubMed Central

The production of different transcripts (transcript heterogeneity) is a feature of many genes that may result in phenotypic variation. Several mechanisms, that occur at both the DNA and RNA level have been shown to contribute to this transcript heterogeneity in mammals, all of which involve either the rearrangement of sequences within a genome or the use of alternative signals in linear, contiguous DNA or RNA. Here we describe tissue-specific repetition of selective exons in transcripts of a rat gene (SA) with a normal exon–intron organization. We conclude that nonlinear mRNA processing can generate tissue-specific transcripts.

Frantz, Simon A.; Thiara, Amrik S.; Lodwick, David; Ng, Leong L.; Eperon, Ian C.; Samani, Nilesh J.



Cell fate conversion by mRNA  

PubMed Central

Recent development of a synthetic mRNA-based technology for efficient reprogramming to pluripotency and cell fate conversion without any modification to the genome has generated great interest among researchers and clinicians alike. It is hoped that this technology could contribute to unmet needs on several fronts of regenerative medicine, including mechanistic study of reprogramming, generation of safe induced pluripotent stem cells suitable for clinical applications, and derivation of desired cell types for cell-replacement therapy. We will discuss the technological advancements made by this synthetic mRNA methodology, its implications, as well as the challenges that lie ahead in the field of regenerative medicine.



Principles of mRNA transport in yeast.  


mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons. PMID:22159587

Heym, Roland Gerhard; Niessing, Dierk



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.  

PubMed Central

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 to 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. Images Fig. 3

Kinter, A L; Bende, S M; Hardy, E C; Jackson, R; Fauci, A S



CD127+CCR5+CD38+++ CD4+ Th1 Effector Cells Are an Early Component of the Primary Immune Response to Vaccinia Virus and Precede Development of Interleukin-2+ Memory CD4+ T Cells  

PubMed Central

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-?)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-? and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.

Zaunders, John J.; Dyer, Wayne B.; Munier, Mee Ling; Ip, Susanna; Liu, Jie; Amyes, Elisabeth; Rawlinson, William; De Rose, Robert; Kent, Stephen J.; Sullivan, John S.; Cooper, David A.; Kelleher, Anthony D.



Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-?  

PubMed Central

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-? were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-? (ChIFN-?) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-? did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

Park, Jeong Ho; Sung, Haan Woo; Yoon, Byung Il



Selective impairment of alpha-interferon-mediated natural killer augmentation in Sj?gren's syndrome: differential effects of alpha-interferon, gamma-interferon, and interleukin 2 on cytolytic activity.  

PubMed Central

Natural killer (NK) function has been shown to be impaired in several autoimmune diseases including Sjögren's syndrome (SS). In the present study, in vitro effects of alpha-interferon (alpha-IFN), gamma-IFN and interleukin 2 (IL-2) on the NK cell activity were examined to analyse the regulatory system of NK-augmentation in patients with SS. The responsiveness of NK cell activity to alpha-IFN was markedly depressed in SS patients compared with normal controls, whereas the responsiveness to gamma-IFN was within normal limits. This is the first demonstration of the selective hyporesponsiveness of NK cell activity to one type of IFN in a certain disease. In addition, the kinetics study of NK-augmentation in normal donors revealed that alpha-IFN enhanced NK cell activity with a faster profile than gamma-IFN. These findings imply substantial differences between the two types of IFN in their mechanisms for enhancing NK cell activity, which deserve attention in evaluating the effects of IFNs. The present study also demonstrated that IL-2 could induce significantly higher levels of NK cell activity than alpha-IFN or gamma-IFN in SS and that this enhancing effect was almost comparable to that in normal controls. Thus, there seem to be multiple regulatory mechanisms for enhancement of NK cell activity, and a portion of the mechanisms may be selectively impaired in certain human diseases such as SS. The selective hyporesponsiveness to alpha-IFN could be relevant to the idea of viral participation in pathogenesis of SS.

Takeda, A; Minato, N; Kano, S



Monocytes and neutrophils as 'bad guys' for the outcome of interleukin-2 with and without histamine in metastatic renal cell carcinoma--results from a randomised phase II trial.  


Histamine (HDC) inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer (NK) and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). We have explored this potential mechanism clinically in two randomised phase II trials in metastatic renal cell carcinoma (mRCC). In parallel with the clinical trial in Denmark (n=63), we obtained serial blood samples and tumour biopsies searching for a potential histamine effect in situ. At baseline and on-treatment weeks 3 and 8, we monitored the 'good guys' (i.e. NK and T cells) and 'bad guys' (i.e. monocytes/macrophages and neutrophils) simultaneously in blood (n=59) and tumour tissue (n=44). Patients with high number of monocytes and neutrophils in peripheral blood had very poor survival, with apparently no benefit from either IL-2 alone or IL-2/HDC treatment. Blood monocytes (r=-0.36, P=0.01) and neutrophils (r=-0.46, P=0.001) were negatively correlated with cytotoxicity, whereas blood NK cells were positively correlated with cytotoxicity (r=0.39, P=0.002). Treatment with IL-2 alone resulted in a significantly higher number of circulating monocytes (P=0.037) and intratumoral macrophages (P=0.005) compared with baseline. In contrast, IL-2/HDC resulted in an unchanged number of circulating monocytes and intratumoral macrophages, and in addition, a significantly increased number of intratumoral CD56(+) NK cells (P=0.008) and CD8(+) T cells (P=0.019) compared with baseline. The study provides evidence that circulating monocytes and neutrophils are powerful negative prognostic factors for IL-2-based immunotherapy and establishes a biological rationale for the potential use of histamine in conjunction with IL-2 in mRCC. PMID:16434984

Donskov, F; Hokland, M; Marcussen, N; Torp Madsen, H H; von der Maase, H



The Majority of Epidermal T Cells in Psoriasis Vulgaris Lesions can Produce Type 1 Cytokines, Interferon-?, Interleukin2, and Tumor Necrosis Factor-?, Defining TC1 (Cytotoxic T Lymphocyte) and TH1 Effector Populations:1 a Type 1 Differentiation Bias is also Measured in Circulating Blood T Cells in Psoriatic Patients  

Microsoft Academic Search

Psoriasis vulgaris is a skin disease potentially mediated by pro-inflammatory cytokines produced by type 1 lesional T cells. The capability of individual T cells to produce these cytokines in lesional skin is not known. In this study we measured the ability of lesional and peripheral blood T cells to produce intracellular interferon-?, tumor necrosis factor-?, interleukin-2, interleukin-4, and interleukin-10 proteins

Lisa M Austin; Maki Ozawa; Toyoko Kikuchi; Ian B Walters; James G Krueger



Renewing the assault on mRNA.  


Mammalian cells dislike double-stranded RNA. They interpret it as a sign of an intruder, and they can unleash a recently discovered defensive mechanism to deal with the problem - they chop the invader into little pieces and use the remnants, called small interfering RNA, to identify and destroy the invader and its progeny. This process, known as RNA interference, may lend itself to new treatments for a wide range of diseases. RNA interference, however, resembles two therapies studied during the 1990s, antisense and ribozymes, in that the gene-silencing target is messenger RNA (mRNA). Is RNA interference really the Next Big Thing - or just a variation on an older but still intriguing theme? PMID:23372488

McCain, Jack



The function of proteins that interact with mRNA  

Microsoft Academic Search

Specific proteins are associated with mRNA in the cytoplasm of eukaryotic cells. The complement of associated proteins depends upon whether the mRNA is an integral component of the polysomal complex being translated, or, alternatively, whether it is part of the non-translated free mRNP fraction. By subjecting cells to ultraviolet irradiation in vivo to cross-link proteins to mRNA, mRNP proteins have

Dawn E. Larson; Bruce H. Sells



General Translational Repression by Activators of mRNA Decapping  

Microsoft Academic Search

Summary Translation and mRNA degradation are affected by a key transition where eukaryotic mRNAs exit transla- tion and assemble an mRNP state that accumulates into processing bodies (P bodies), cytoplasmic sites of mRNA degradation containing nontranslating mRNAs, and mRNA degradation machinery. We identify the decapping activators Dhh1p and Pat1p as functioning as translational repressors and facilitators of P body formation.

Jeff Coller; Roy Parker



Regression of bladder tumors in mice treated with interleukin 2 gene- modified tumor cells [published erratum appears in J Exp Med 1993 Jun 1;177(6):following 1831  

PubMed Central

This study explored the use of interleukin 2 (IL-2) and interferon gamma (IFN-gamma) gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner similar to its human counterpart. Using retroviral vectors, the human IL-2 and mouse IFN- gamma genes were introduced and expressed in MBT-2 cells. The tumor- forming capacity of the cytokine gene-modified MBT-2 cells was significantly impaired, since no tumors formed in mice injected intradermally with either IL-2- or IFN-gamma-secreting cells, using cell doses far exceeding the minimal tumorigenic dose of parental MBT-2 cells. Furthermore, mice that rejected the IL-2- or IFN-gamma-secreting tumor cells became highly resistant to a subsequent challenge with parental MBT-2 cells, but not to 38C13 cells, a B cell lymphoma of the same genetic background. To approximate the conditions as closely as possible to the conditions prevailing in the cancer patient, inactivated cytokine-secreting cells were used to treat animals bearing tumors established by orthotopic implantation of MBT-2 cells into the bladder wall of the animal. Treatment of mice carrying a significant tumor burden with IL-2-secreting MBT-2 cells had a significant inhibitory effect on tumor progression with extended survival. Moreover, in 60% of the mice the tumor regressed completely and the animals remained alive and free of detectable tumor for the duration of the observation period. Treatment of tumor-bearing animals with IL-2- secreting MBT-2 cells was superior to the use of cisplatin, a chemotherapeutic agent used in the treatment of bladder cancer. The therapeutic effect of IFN-gamma-secreting cells was minimal and treatment with unmodified MBT-2 cells had no effect on tumor growth or survival, showing that the parental MBT-2 cells were nonimmunogenic in this experimental setting. Most importantly, mice that exhibited complete tumor regression after treatment with IL-2-secreting MBT-2 cells became resistant to a subsequent challenge with a highly tumorigenic dose of parental MBT-2 cells, indicating that long-term immunological memory was established in the "cured" mice.



Estradiol regulates estrogen receptor mRNA stability.  


Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA. PMID:9719445

Saceda, M; Lindsey, R K; Solomon, H; Angeloni, S V; Martin, M B



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.  


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

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



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


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

Custodia-Lora, Noemí; Callard, Ian P



Discordant Protein and mRNA Expression in Lung Adenocarcinomas  

Microsoft Academic Search

The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abun- dance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine

Guoan Chen; Tarek G. Gharib; Chiang-Ching Huang; Jeremy M. G. Taylor; David E. Misek; Sharon L. R. Kardia; Thomas J. Giordano; Mark D. Iannettoni; Mark B. Orringer; Samir M. Hanash; David G. Beer



RNA Probe for Detecting c-fes mRNA.  

National Technical Information Service (NTIS)

A recombinant plasmid and an RNA sequence expressed by said plasmid are described. The RNA sequence hybridizes specifically with human c-fes mRNA. It is, therefore, an object of the present invention to provide a kit for the detection of c-fes mRNA in bio...

R. Glazer



[Expression of IRP2 mRNA, TfR mRNA and Fn mRNA in HL-60 cells].  


To explore the mechanism of iron metabolism and its regulation as well as the roles of IRP(2) in ion metabolism of HL-60 cells, HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, which was treated with ferric chloride (FeCl(3)) or deferoxamine (DFO). The cells were harvested at 12, 24 and 48 hours of proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription (RT), and relative expression levels of IRP(2) mRNA, Fn mRNA and TfR mRNA were determined by RT-PCR. The results showed at follows: (1) the level of IRP(2) mRNA remained constant in all cells, whether or not treated with DFO or FeCl(3). However, the expression of IRP(2) mRNA decreased when the time of cell culture was prolonged. There was no significant difference between groups (F(B-S) = 1.199, P > 0.05), but there was significant difference among the different time culture (F(W-S) = 43.418, P < 0.01). (2) Cells which treated neither with DFO nor ferri chloride showed significant difference from the control (F(W-S) = 7.184, F(B-S) = 113.926; P < 0.01). The level of TfR mRNA increased in the cells treated with DFO. Surprisingly, when cells treated with FeCl(3), there was not decline of TfR mRNA expression, but it increased lightly at 12 hours and peaked at 24 hours and declined drastically at 48 hours. (3) The level of Fn mRNA in the cells treated with FeCl(3) was approximately 2-fold as the control cells. In contrast with the control cells, there was significant difference (P < 0.05). The level of Fn mRNA of the cells treated with DFO had little change. As compared with the control cells, no significant difference was seen (P > 0.05). (4) There was not any significant correlation between IRP(2) mRNA and TfR mRNA or Fn mRNA in HL-60 cells (r = -0.005; r = 0.074; P > 0.05). It is concluded that (1) IRP(2) may regulate the iron metabolism in HL-60 cells by altering amounts of the IRP(2) 3.7- or 6.4-kb mRNA at the transcriptional level, or by IRP(2) degradation at the post transcriptional level. (2) Both of Fn mRNA and TfR mRNA participated, more or less, in the iron metabolism in HL-60 cells. PMID:16129039

Liu, Yu-Feng; Zhang, Chuan-Xin; Zeng, Li



Tyrosine aminotransferase mRNA turnover in CHO cells  

SciTech Connect

An expression vector containing a minigene designed for precise expression of the mRNA and the protein of rat tyrosine aminotransferase (TAT), ordinarily only expressed in liver, was constructed. This minigene was placed under control of the LTR promoter of murine leukemia virus and was cotransfected into CHO cells deficient in dihydrofolate reductase (DHFR) with another vector (pSV2dhfr) encoding DHFR. Upon methotrexate selection and further amplification, cell lines which produce large amounts of TAT mRNA and the enzyme have been isolated. Northern blots exhibited the expected 2.4 kb band of TAT mRNA, and S1-nuclease protection assay established that the mRNA was correctly initiated and terminated. When transcription was blocked with actinomycin, the 2.4 kb TAT mRNA in CHO cell lines was degraded with half-life ranging 1.5 to 2.5 hr, a rate similar to that found in liver or hepatoma cells. Furthermore, the rapid turnover of TAT mRNA in CHO cells could be completely stabilized by cyclohexamide as observed previously in rat liver. With their high content of TAT mRNA exhibiting normal turnover properties, the CHO cell lines established should be useful for analysis of the molecular mechanisms of intracellular turnover of this labile message.

Lee, K.L.; Yang, D.M.; Makkinje, A.; Ch'ang, L.Y. (Oak Ridge National Lab., TN (United States) Univ. of Tennessee, Oak Ridge (United States))



Imaging Native ?-Actin mRNA in Motile Fibroblasts  

PubMed Central

Nuclease-resistant, cytoplasmically resident molecular beacons were used to specifically label ?-actin mRNA in living and motile chicken embryonic fibroblasts. ?-actin mRNA signals were most abundant in active lamellipodia, which are protrusions that cells extend to adhere to surfaces. Time-lapse images show that the immediate sources of ?-actin mRNA for nascent lamellipodia are adjacent older protrusions. During the development of this method, we observed that conventional molecular beacons are rapidly sequestered in cell nuclei, leaving little time for them to find and bind to their cytoplasmic mRNA targets. By linking molecular beacons to a protein that tends to stay within the cytoplasm, nuclear sequestration was prevented, enabling cytoplasmic mRNAs to be detected and imaged. Probing ?-actin mRNA with these cytoplasmically resident molecular beacons did not affect the motility of the fibroblasts. Furthermore, mRNAs bound to these probes undergo translation within the cell. The use of cytoplasmically resident molecular beacons will enable further studies of the mechanism of ?-actin mRNA localization, and will be useful for understanding the dynamics of mRNA distribution in other living cells.

Tyagi, Sanjay; Alsmadi, Osama



Phenotypic and functional properties of murine thymocytes. I. Precursors of cytolytic T lymphocytes and interleukin 2-producing cells are all contained within a subpopulation of "mature" thymocytes as analyzed by monoclonal antibodies and flow microfluorometry  

PubMed Central

The correlation between surface phenotype and function in subpopulations of murine thymocytes has been investigated using flow microfluorometry (FMF). C57BL/6 thymocytes stained with monoclonal antibodies directed against Lyt-2, H-2K(b), and Thy-l.2 and passed on an FACS II flow cytometer could be resolved into at least four distinct subpopulations on the basis of fluorescence and forward light scatter (FLS) measurements. (a) Medium-sized Lyt-2(+) cells that stained strongly with H-2K(b) and weakly with Thy-l.2 (5 percent of total cells); (b) medium-sized Lyt-2(-) cells with other properties as in (a) (10 percent); (c) small Lyt-2(+) cells that stained weakly with H-2K(b) and strongly with Thy-l.2 (60 percent); and (d) large Lyt-2(+) cells that stained weakly with H-2K(b) and very strongly with Thy- 1.2 (23 percent). Cortisone-resistant thymocytes (CRT) were found to correspond phenotypically to populations (a) and (b). The distribution of cytolytic T lymphocyte precursors (CTL-P) directed against H-2(d) alloantigens in subpopulations of C57BL/6 thymocytes that had been sorted according to the phenotypic criteria described above was then investigated. CTL-P in sorted and control populations were quantitated by limiting dilution analysis of mixed leukocyte microcultures established in an excess of interleukin 2 (IL-2). These studies established that all thymus CTL-P could be quantitatively recovered in a subpopulation of cells that was cortisone-resistant, medium-sized, Lyt-2(+), H-2K(b+), and weakly stained with Thy-l.2. In parallel studies, the production of IL-2 by subpopulations of C57BL/6 thymocytes was quantitatively assessed using a recently developed sensitive microassay system. Graded numbers of sorted or control thymocytes were stimulated with irradiated T cell-depleted allogeneic cells and assayed for their ability to support the growth of an IL-2-dependent cytolytic T lymphocyte clone. Using this method, IL-2 production was found to reside entirely in a subpopulation of cortisone-resistant, medium-sized Lyt-2(-) thymocytes. Further phenotypic analysis of this subpopulation of cells indicated that it was homogeneously H-2K(b+) and weakly staining with Thy- 1.2. Taken together with the CTL-P results, these data directly demonstrate that a subpopulation of thymocytes with a mature phenotype (i.e., cortisone- resistant, medium-sized, H-2K(b+), and weakly staining with Thy-l.2) accounts for all the functional activity in the thymus. Reasons for the apparent discrepancy between these results and other recent studies will be discussed.

Ceredig, R; Glasebrook, AL; MacDonald, HR



Production of porcine interleukin-2 and its biological and antigenic relationships with human interleukin-2.  


A lymphokine obtained from PHA-stimulated porcine blood leukocytes was found to induce the growth of IL2-dependent murine cells: this factor was therefore considered as the porcine lymphokine analogous to IL2. PHA was found to induce higher amounts of IL2 than Con A. Preculture of porcine PBL or addition of indomethacin enhanced IL2 production, suggesting the existence of a prostaglandin-mediated inhibition of IL2 production in fresh PBL cultures. Porcine IL2 could also promote the growth of activated porcine cells whereas it was inactive on human lymphoblastoid cells. A neutralizing polyclonal anti-human IL2 antiserum was also unable to neutralize porcine IL2. These results indicate therefore, that no biological and antigenic cross-reactivities are demonstrable between human and porcine IL2. PMID:3876277

Charley, B; Petit, E; Leclerc, C; Stefanos, S



Signaling pathways that control mRNA turnover.  


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

Thapar, Roopa; Denmon, Andria P



Localization and anchoring of mRNA in budding yeast  

Microsoft Academic Search

as a means to sequester proteins. Signals within the 3 untranslated region (3UTR) facilitate mRNA localization by both actin and microtubule cytoskeletal systems. Recently, an mRNA in the yeast Saccharomyces cerevisiae, ASH1, was shown to coalesce into a discrete particle that is maintained at the bud tip. Mutations in five genes, SHE1-SHE5, cause defects in particle formation and\\/or localization of

Dale L. Beach; E. D. Salmon; Kerry Bloom



Localization and anchoring of mRNA in budding yeast  

Microsoft Academic Search

Background: Eukaryotic cells localize selected mRNAs to a region of the cell as a means to sequester proteins. Signals within the 3? untranslated region (3? UTR) facilitate mRNA localization by both actin and microtubule cytoskeletal systems. Recently, an mRNA in the yeast Saccharomyces cerevisiae, ASH1, was shown to coalesce into a discrete particle that is maintained at the bud tip.

Dale L. Beach; E. D. Salmon; Kerry Bloom



Directional mRNA transport in eukaryotes: lessons from yeast  

Microsoft Academic Search

.  In eukaryotes, developmental processes and cell differentiation, as well as basic cellular functions require the propagation\\u000a of information in an asymmetric manner. Localization of mRNA is a key mechanism to establish asymmetric cell fate. The first\\u000a part of this review provides an overview of our current knowledge of motor protein-dependent mRNA transport in eukaryotes.\\u000a The second part provides a more

M. Müller; A. Heuck; D. Niessing



The terminal structures of feather keratin mRNA  

Microsoft Academic Search

Terminal labeling of embryonic feather keratin mRNA with [3H]KBH4 followed by digestion with ribonuclease T1 and T2, alkaline phosphatase, snake venom phosphodiesterase, and nucleotide pyrophosphatase and analysis of the products by high voltage paper electrophoresis, indicated the presence of the sequence m7G(5')ppp(5')N at the 5'-end of the mRNA. Ribonuclease T1 and A digests of the terminally labeled, and also of

C. Phillip Morris; George E. Rogers



Polyadenylation accelerates degradation of chloroplast mRNA.  

PubMed Central

The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation. Images

Kudla, J; Hayes, R; Gruissem, W



Mechanisms of endonuclease-mediated mRNA decay  

PubMed Central

Endonuclease cleavage was one of the first identified mechanisms of mRNA decay but until recently it was thought to play a minor role to the better-known processes of deadenylation, decapping and exonuclease-catalyzed decay. Most of the early examples of endonuclease decay came from studies of a particular mRNA whose turnover changed in response to hormone, cytokine, developmental or nutritional stimuli. Only a few of these examples of endonuclease-mediated mRNA decay progressed to the point where the enzyme responsible for the initiating event was identified and studied in any detail. The discovery of microRNAs and RISC-catalyzed endonuclease cleavage followed by the identification of PIN (pilT N-terminal) domains that impart endonuclease activity to a number of the proteins involved in mRNA decay has led to a resurgence of interest in endonuclease-mediated mRNA decay. PIN domains show no substrate selectivity, and their involvement in a number of decay pathways highlights a recurring theme that the context in which an endonuclease functions is a primary factor in determining whether any given mRNA will be targeted for decay by this or the default exonuclease-mediated decay processes.

Schoenberg, Daniel R.



Cytoplasmic organelles on the road to mRNA decay.  


Localization of both mRNAs and mRNA decay factors to internal membranes of eukaryotic cells provides a means of coordinately regulating mRNAs with common functions as well as coupling organelle function to mRNA turnover. The classic mechanism of mRNA localization to membranes is the signal sequence-dependent targeting of mRNAs encoding membrane and secreted proteins to the cytoplasmic surface of the endoplasmic reticulum. More recently, however, mRNAs encoding proteins with cytosolic or nuclear functions have been found associated with various organelles, in many cases through unknown mechanisms. Furthermore, there are several types of RNA granules, many of which are sites of mRNA degradation; these are frequently found associated with membrane-bound organelles such as endosomes and mitochondria. In this review we summarize recent findings that link organelle function and mRNA localization to mRNA decay. This article is part of a Special Issue entitled: RNA Decay mechanisms. PMID:23337852

Weil, Dominique; Hollien, Julie



Platelet mRNA: the meaning behind the message  

PubMed Central

Purpose of review It is now well appreciated that megakaryocytes invest platelets with a diverse repertoire of messenger RNAs (mRNAs), which are competent for translation. Herein we describe what is currently known regarding the expression, function, and clinical significance of mRNAs in platelets. Recent findings Although mRNA was detected in platelets nearly 30 years ago, we are only beginning to understand the roles of mRNA in platelet biology and human disease. Recent studies have shown that megakaryocytes specifically sort, rather than randomly transfer, mRNA to platelets during thrombopoiesis. As a result, platelets are released into the circulation with thousands of mRNAs. The emergence of next-generation RNA sequencing has demonstrated that platelet mRNAs possess classic structural features, which include untranslated regions and open reading frames. There is also growing evidence that platelet mRNA expression patterns are altered in human disease. Summary Intense investigation of platelet mRNA has shed considerable light on predicted functions of platelets and identified previously unrecognized attributes of platelets. Lessons learned from platelet mRNA is presented in this review.

Rowley, Jesse W.; Schwertz, Hansjorg; Weyrich, Andrew S.



Making the message clear: visualizing mRNA localization  

PubMed Central

Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology. To explore the mechanisms involved, considerable effort has been spent in establishing new and improved methods for visualizing mRNA. Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms. In addition to increased ease and specificity of detection in fixed tissue, in situ hybridization methods now enable examination of mRNA distribution at the ultrastructural level with electron microscopy. Most significantly, methods for following the movement of mRNA in living cells are now in widespread use. These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components. These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization.

Weil, Timothy T.; Parton, Richard M.; Davis, Ilan



Establishment of a monitoring system to detect inhibition of mRNA processing.  


A number of proteins complete mRNA processing in the nucleus, thus, inhibitor of mRNA processing is worth finding to analyze the mechanism of mRNA maturation in detail. Here, we established a monitoring system for mRNA processing using a test compound, spliceostatin A (SSA), which inhibits mRNA splicing. This system should serve to facilitate the discovery of novel compounds from natural resources that inhibit mRNA processing. PMID:22790958

Fujita, Ken-ichi; Okamura, Masumi; Nishimoto, Sachiko; Kurihara, Tomoya; Momma, Keiko; Miyamae, Yusaku; Kambe, Taiho; Nagao, Masaya; Narita, Hiroshi; Masuda, Seiji



Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability  

Microsoft Academic Search

BACKGROUND: Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels.

Chris Cheadle; Jinshui Fan; Yoon S Cho-Chung; Thomas Werner; Jill Ray; Lana Do; Myriam Gorospe; Kevin G Becker



Destabilization of TNF-? mRNA by Rapamycin  

PubMed Central

Stimulation of mast cells through the high affinity IgE receptor (Fc?RI) induces degranulation, lipid mediator release, and cytokine secretion leading to allergic reactions. Although various signaling pathways have been characterized to be involved in the Fc?RI-mediated responses, little is known about the precious mechanism for the expression of tumor necrosis factor-? (TNF-?) in mast cells. Here, we report that rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), reduces the expression of TNF-? in rat basophilic leukemia (RBL-2H3) cells. IgE or specific antigen stimulation of RBL-2H3 cells increases the expression of TNF-? and activates various signaling molecules including S6K1, Akt and p38 MAPK. Rapamycin specifically inhibits antigen-induced TNF-? mRNA level, while other kinase inhibitors have no effect on TNF-? mRNA level. These data indicate that mTOR signaling pathway is the main regulation mechanism for antigen-induced TNF-? expression. TNF-? mRNA stability analysis using reporter construct containing TNF-? adenylate/uridylate-rich elements (AREs) shows that rapamycin destabilizes TNF-? mRNA via regulating the AU-rich element of TNF-? mRNA. The antigen-induced activation of S6K1 is inhibited by specific kinase inhibitors including mTOR, PI3K, PKC and Ca2+chelator inhibitor, while TNF-? mRNA level is reduced only by rapamycin treatment. These data suggest that the effects of rapamycin on the expression of TNF-? mRNA are not mediated by S6K1 but regulated by mTOR. Taken together, our results reveal that mTOR signaling pathway is a novel regulation mechanism for antigen-induced TNF-? expression in RBL-2H3 cells.

Park, Jong-Woo; Jeon, Ye Ji; Lee, Jae Cheol; Ahn, So Ra; Ha, Shin Won; Bang, So Young; Park, Eun Kyung; Yi, Sang Ah; Lee, Min Gyu; Han, Jeung-Whan



Cytokines in chronic inflammatory arthritis. I. Failure to detect T cell lymphokines (interleukin 2 and interleukin 3) and presence of macrophage colony-stimulating factor (CSF-1) and a novel mast cell growth factor in rheumatoid synovitis  

PubMed Central

Because previous studies showed low levels of IFN-gamma in rheumatoid arthritis (RA) synovial fluid (SF) and synovial tissue (ST) explant supernatants, we assayed RA SF and ST for IL-2 and IL-3-like activity. Using an IL-2 dependent murine CTLL line, 6 of 14 RA SF caused increased thymidine uptake (greater than three times control). The activity was distinct from IL-2 because it was not blocked by antibody to IL-2-R. In addition, IL-2 was not detected (less than 50 pg/ml) in 16 joint samples using an ELISA. Multi-colony-stimulating factor (CSF) activity was measured using two assays that can detect murine IL-3 (mast cell proliferation, and bone marrow CSF). In the mast cell assay, [3H]TdR uptake was 493 +/- 67 cpm for medium, 2,910 +/- 329 cpm in the presence of RA SF (p less than 0.001), 1,246 +/- 156 cpm in the presence of SF from patients with seronegative spondyloarthropathies (p less than 0.001), and 736 +/- 100 cpm in the presence of osteoarthritis SF (p greater than 0.1). In the CSF assay, four of five RA SF and five of five RA ST induced colony formation from bone marrow nonadherent cells. Macrophage colonies were most common, although mixed colonies and granulocytes were occasionally observed. The multi-CSF activity in RA is not due to IL-3 since human rIL-3 was not active in either murine assay, and IL-3 mRNA was not detected in RA synovium. Sephadex column chromatography of RA SF revealed that the mast cell growth factor (approximately 6 x 10(3) mol wt) and the CSF (approximately 40 and 100 x 10(3) mol wt) are distinct. The colony-stimulating aspect of the "IL- 3-like" activity in RA SF is likely due to CSF-1 because it is the appropriate mol wt and because the activity was neutralized by specific anti-CSF-1 antibody. Finally, an RIA detected 1.6-25 ng/ml of CSF-1 in RA SF and ST and CSF-1 mRNA was detected in four of five RA synovial tissue samples tested.



Endothelial cell expression of testican mRNA.  


By screening random cDNAs from a continuous vascular endothelial cell line, EA.hy926, we identified a 5 kb mRNA that is expressed at hig