In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2) on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter (PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6 x 10(5) u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.
Animals ; Chick Embryo ; Chickens ; Fowlpox virus ; genetics ; Interleukin-2 ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; pharmacology

OBJECTIVETo study the role of MEKK2 on the production of IL-2 in Jurkat cells stimulated by PHA/anti-CD28 antibody.

METHODSThe MEKK2 and JNK kinase activities were measured in both dominant negative MEKK2 Jurkat (dnMEKK2 Jurkat) cells and parental Jurkat cells. The AP(1) and IL-2 promotor activities were measured by luciferase activity assay. The IL-2 mRNA and protein were detected by RT-PCR and Western blot.

RESULTSAfter stimulation by PHA/anti-CD28, JNK was activated in parental Jurkat cells but not in dnMEKK2 Jurkat cells. The luciferase report gene activities of AP1 and IL-2 promotors were increased by 4- and 5-folds in parental cells whereas only by 1 fold in dnMEKK2 Jurkat cells. The level of IL-2 mRNA and IL-2 protein were increased in parental Jurkat cells but not in dnMEKK2 Jurkat cells.

CONCLUSIONMEKK2 plays an important role on the production of IL-2 in Jurkat cell stimulated with PHA/anti-CD28 antibody. It is a potential drug target for the treatment of GVHD and autoimmune disease.

Humans ; Interleukin-2 ; biosynthesis ; genetics ; Jurkat Cells ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Kinase Kinase 2 ; metabolism ; physiology

OBJECTIVETo optimize the culture condition of engineered E.coli to improve its expression efficiency of hIL-2-mGM-CSF protein.

METHODSAccording to an orthogonal Latin square experiment design, the effects of the culture medium, temperature and IPTG concentration at different levels on the efficiency of the engineered E. coli were evaluated for its expression of hIL-2-mGM-CSF protein. The results of SDS-PAGE were analyzed with software and the culture conditions derived from the experimental results were tested in independent cultures. The optimal culture condition was used in three large-scale cultures and the results were compared with that of routine cultures.

RESULTSThe cultures with TH broth yielded higher relative expression quantity of the target protein than those with 2 x YT and LB medium. Compared with the induction temperature at 37 degrees C, induction at 42 degrees C significantly improved the expression efficiency of the target protein. IPTG at the concentration as low as 0.3 mmol/L produced better effect than 1.0 mmol/L IPTG. Statistical analysis suggested that the optimized culture conditions could obviously improve the expression efficiency of the target protein. Large scale cultures with the optimized culture condition resulted in a 5-fold improvement of the relative expression quantity of the protein, which accounted for over 29% of total bacterial protein.

CONCLUSIONThe optimized culture condition of the engineered E. coli can remarkably increase the expression efficiency of hIL-2-mGM-CSF, which may facilitate the subsequent purification and functional study of the protein.

Culture Media ; Escherichia coli ; genetics ; growth & development ; Genetic Engineering ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Interleukin-2 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic

Interleukin-2 (IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differentiation of several hematopoietic cell types. Clinical studies using IL-2 in the treatment of AIDS have been encouraging, due to its critical role as a proliferative signal for activated T-lymphocytes. IL-2 has also undergone trials in the treatment of several types of cancer, based on its stimulation of cytotoxic, antitumor cells. Today, human IL-2 is produced completely by genetically engineered method, and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. In the former study, recombinant human IL-2 usually comes from E. coli, in this paper the mutant IL-2 was successfully expressed and purified in Pichia pastoris for the first time. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. Expression conditions of human mutant interleukin-2(the codon for cysteine-125 of human IL-2 with alanine; the codon for leucine-18 with methionine; the codon for leucine-19 with serine) in the recombinant Pichia pastoris strain were optimized via test of some factors such as the rate of aeration, the inductive duration, the initial pH and the concentration of methanol. The results from tests showed that the most important parameter for efficient expression of interleukin-2 in recombinant Pichia pastoris strain is adequate aeration during methanol induction, and the optimum inductive condition for interleukin-2 expression was: more than 80% aeration, 2 days for induction, the initial pH of 6.0, the final methanol concentration of 1.0%. With this condition, the expressed IL-2 was secreted into fermentation broth and reached a yield of 30%, approximately 200 mg/L. Expressed interleutin-2 (MvIL-2) was isolated and purified by centrifugation, millipore filtration to concentration, Econo-PacS strongly acidic cation exchanger cartridge and molecular sieve chromatography and the yield of MvIL-2 was 27%. MvIL-2 was purified to electrophoretic purity by SDS-PAGE and only one peak being loaded on HPLC. Purified MvIL-2 protein had stimulating activity similar to the wild type of IL-2 as assayed by IL-2-dependent CTLL-2 cells. However, the stability of MvIL-2 was superior than that of IL-2 at different temperatures. The activity of obtained MvIL-2 was 4 - 5 times of the wild type of IL-2, So MvIL-2 had an advantage over wild type of rhIL-2 in storage stability and activity.
Fermentation ; Humans ; Interleukin-2 ; biosynthesis ; genetics ; Mutant Proteins ; biosynthesis ; Mutation ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.
4-1BB Ligand ; biosynthesis ; genetics ; Apoptosis ; genetics ; Cell Line ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Recombinant Proteins ; biosynthesis ; genetics

To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in preventing graft-versus-host disease (GVHD), the CD34+ cells transfected with FasL or not, used as stimulus cells, were mixed with allo-antigen specific T lymphocytes in presence or absence of IFN-gamma and IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry (FCM). The affects of these two cytokines on CD34+ cells in the graft were also compared. The ratio of apoptosis of T cells was 12.1+/-1.5% when CD34+ cells transfected with FasL was used as stimulus cells, much higher than that of CD34+ cells non-transfected (3.2+/-1.1%, P<0.01). And in presence of IFN-gamma or IL-2, the ratio reached 20.1+/-2.3%, 17.6+/-1.3% respectively (P<0.01). However, IFN-gamma up-regulated Fas expression of CD34+ cells and increased the sensibility of CD34+ cells to soluble FasL (sFasL); IL-2 showed no such effect. It is possible to induce apoptosis of the allo-antigen specific T cells of grafts activated by allo-antigen by exogenous Fas ligand expressed on recipient cells and this might provide a new approach for preventing GVHD and IL-2 may be more suitable for clinical application.
Antigens, CD34 ; biosynthesis ; immunology ; Apoptosis ; Cytotoxicity, Immunologic ; DNA, Complementary ; genetics ; Fas Ligand Protein ; Graft vs Host Disease ; prevention & control ; Interferon-gamma ; biosynthesis ; immunology ; Interleukin-2 ; biosynthesis ; immunology ; Membrane Glycoproteins ; biosynthesis ; immunology ; T-Lymphocytes ; cytology ; physiology ; fas Receptor ; biosynthesis ; immunology

The study was purposed to investigate the effects of humanized recombined CD25 monoclonal antibody (rhCD25MAb) on activation and proliferation of T lymphocytes in vitro. Peripheral blood mononuclear cells (PBMNC) were incubated with phytohemagglutinin (PHA). Before or after T cell activation, the cells were cultured with or without rhCD25MAb or cyclosporine A (CsA) in vitro. After 72 hours incubation, the proliferation of lymphocytes was analyzed by MTT assay. The expression of CD3 and CD25 antigens on T lymphocytes were detected by flow cytometry. The levels of sIL-2R in the supernatants were determined with ELISA. The results showed that both rhCD25MAb and CsA could inhibit the proliferation of T lymphocytes significantly in concentration-dependent manner and CsA was more efficient than rhCD25MAb. Both rhCD25MAb and CsA could also decrease the levels of sIL-2R in the supernatant and inhibit the expression of CD25 antigen on T lymphocytes. The level of sIL-2R and the expression of CD25 on T lymphocytes decreases more profoundly in rhCD25MAb group. It is concluded that rhCD25MAb shows strong immunosuppressive activity both before and after T cell activation, suggesting that this agent may be useful in not only prophylaxis but also the treatment of acute graft-versus-host disease.
Antibodies, Monoclonal ; pharmacology ; CD3 Complex ; biosynthesis ; genetics ; Cell Proliferation ; Cells, Cultured ; Cyclosporine ; pharmacology ; Humans ; Immunosuppressive Agents ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; biosynthesis ; genetics ; immunology ; Lymphocyte Activation ; immunology ; Receptors, Interleukin-2 ; analysis ; Recombinant Proteins ; immunology ; pharmacology ; T-Lymphocytes ; immunology

The relationship between T cell immunoglobulin domain and mucin domain protein 3 (Tim-3)/Galectin (Gal)-9 pathway and recurrent spontaneous abortion (RSA) was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin (IL)-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05), but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05). It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.
Abortion, Spontaneous ; metabolism ; pathology ; Adolescent ; Adult ; Chorionic Villi ; metabolism ; pathology ; Female ; Galectins ; biosynthesis ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; Membrane Proteins ; biosynthesis ; Pregnancy ; Pregnancy Proteins ; biosynthesis ; Up-Regulation

AIMTo study the effects of ginsenoside-Ro on cell proliferation and cytokine production in murine splenocytes.

METHODSThe effect of ginsenoside-Ro on murine splenocytes proliferation was studied using [3H] thymidine incorporation assay. Effects of ginsenoside-Ro on the production of cytokines interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) from murine splenocytes were detected by ELISA method. Effects of ginsenoside-Ro on mRNA level of Th1 cytokine IFN-gamma and Th2 cytokine IL-4 were evaluated by reverse transcription polymerase chain reaction (RT-PCR) analysis.

RESULTSGinsenoside-Ro showed no mitogenic effect on unstimulated murine splenocytes. It enhanced the proliferation of Con A-induced murine splenocytes and the production of IL-2 at concentrations of 1-10 micromol x L(-1). Moreover, ginsenoside-Ro increased the production and expression of Th2 cytokine IL-4 and decreased the production and expression of Th1 cytokine IFN-gamma in Con A-induced murine splenocytes at concentrations of 2-10 micromol x L(-1).

CONCLUSIONGinsenoside-Ro showed immunomodulatory effects by regulating the production and expression of Th1/Th2 cytokines in murine splenocytes.

Animals ; Cell Proliferation ; drug effects ; Ginsenosides ; isolation & purification ; pharmacology ; Interferon-gamma ; biosynthesis ; genetics ; Interleukin-2 ; metabolism ; Interleukin-4 ; biosynthesis ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Spleen ; cytology ; metabolism

OBJECTIVETo investigate whether GM-CSF can enhance the antiviral effect of HBsAg DNA vaccine.

METHODSDivided animals into 8 groups. Group A: pcDNA3.1-S 100microg; Group B: pcDNA3.1-GM-CSF-S 100microg; Group C: pcDNA3.1-S-GM-CSF 100microg; Group D: pcDNA3.1-S 50microg + pcDNA3.1-GM-CSF 50microg; Group E: pcDNA3.1-GM-CSF 100microg; Group F: recombinant HBsAg vaccine 1microg; Group G: pcDNA3.1,100microg; Group H: PBS 100microl. Serum HBsAg level and concentration of IL-2, IL-4 and IFN-gamma were examined using commercial ELISA kit. The [3H] thymidine incorporation into DNA of Spleen cells was measured; HBsAg expression of hepatocytes from HBV-transgenic mice was assessed using immunohistochemical analysis.

RESULTSSerum HBsAg level was lower and concentration of IL-2, IFN-gamma and SI was higher in mice immunized with pcDNA3.1-GM-CSF-S than those from mice immunized with pcDNA3.1-S and other groups (F=11.262, P<0.01, F=8.147, P<0.01, F=62.275, P<0.01, F=116.28, P<0.01. Less Hepatic HBsAg expression and decline of pcDNA3.1-GM-CSF-S of mice immunized with pcDNA3 were observed in comparison with control groups (F=41.439, P<0.01). Liver histological analysis showed no evidence of necrosis or inflammation in various groups.

CONCLUSIONThe plasmid co expressing GM-CSF and HBsAg could improve HBsAg-specific humoral and cellular immune responses induced by plasmid encoding HBsAg alone and enhance HBsAg DNA vaccine antivirus effect.

Animals ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B Vaccines ; immunology ; Interferon-gamma ; biosynthesis ; Interleukin-2 ; biosynthesis ; Interleukin-4 ; biosynthesis ; Lymphocyte Activation ; Male ; Mice ; Mice, Transgenic ; Plasmids ; Vaccines, DNA ; immunology