The porcine interferon-gamma (PoIFN-gamma) gene, in which the sequence encoding signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-alpha-PoIFN-gamma was then transformed into Pichia pastoris GS115 cells by electroporation and stable multicopy recombinant Pichia pastoris strains were selected by G418 resistance. Two recombinants of multiple inserts were obtained. SDS-PAGE and Western blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant PoIFN-gamma, 17kD and 23kD proteins, were secreted into the culture medium. Target proteins, 60% of total proteins, were obtained in the culture medium at the concentration of 108 mg/L. This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris.
Animals ; Electroporation ; Interferon-gamma ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; genetics

This paper aims to explore the effects of chicken interferon-γ (ChIFN-γ) and interleukin-2 (ChIL-2) on type 1 helper (Th1) T lymphocyte differentiation. To be specific, ChIFN-γ and ChIL-2 were first expressed in Escherichia coli competent cells and then purified by Ni-NTA affinity chromatography. Different concentration of ChIFN-γ and ChIL-2 were employed to stimulate the lymphocytes in chicken peripheral blood which had been activated by concanavalin A (Con A), and the mRNA levels of cytokines related to Th1 cell differentiation were detected by real-time quantitative PCR (RT-qPCR). The results showed that both ChIFN-γ and ChIL-2 can significantly up-regulate mRNA levels of cytokines related to Th1 cell differentiation and the optimal concentration was 12.5 μg/mL and 25.0 μg/mL, respectively. In addition, specific-pathogen-free (SPF) chickens were immunized with ChIL-2 or ChIFN-γ together with H9N2 vaccine, or H9N2 vaccine alone by oral administration or intramuscular injection, respectively. The mRNA levels of cytokines related to Th1 cell differentiation were detected after immunization. The results showed that ChIFN-γ and ChIL-2 significantly up-regulated the mRNA levels of cytokines related to Th1 cell differentiation induced by H9N2 vaccine compared with H9N2 vaccine alone, and that the intramuscular injection was better than oral administration. In this study, we verified that ChIFN-γ and ChIL-2 can significantly enhance mRNA levels of cytokines related to Th1 cell differentiation induced by ConA or H9N2 vaccine in vitro and in vivo. The results of this study can lay a theoretical basis for using ChIFN-γ and ChIL-2 as vaccine adjuvants.
Animals ; Cell Differentiation ; Chickens ; Concanavalin A ; Cytokines/genetics* ; Influenza A Virus, H9N2 Subtype/genetics* ; Interferon-gamma/metabolism* ; Interleukin-2/genetics* ; RNA, Messenger

This study examined the effects of Bangdeyun on the expressions of nuclear factor-kappaB (NF-kappaB), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the endometrium of mice with embryo implantation dysfunction (EID) during the implantation time (namely on pregnancy day 5, 6, 7 and 8) and explored the local immune regulatory effects of Bangdeyun. The gestational mice were randomly divided into normal group, model group and Bangdeyun-treated group. EID models of mice were established by using indomethacin. The endometrial expression of NF-kappaB was detected by immunohistochemistry and Western blotting. IFN-gamma and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that in the normal group, NF-kappaB and IFN-gamma were weakly expressed and IL-10 was strongly expressed in the endometrium during the whole implantation period. In the model group, the expressions of NF-kappaB and IFN-gamma were increased on pregnancy day 5, 6 and 7, and IL-10 expression decreased during the whole implantation time when compared with those in the normal group (P<0.01 for all). In the Bangdeyun-treated group, little amount of NF-kappaB and IFN-gamma was expressed and IL-10 expression was strong, much the way they were expressed in the normal group (P>0.05). The expressions of NF-kappaB and IFN-gamma were much lower in the Bangdeyun-treated group than those in the model group on pregnancy day 5, 6 and 7 (P<0.01 for all), while the expression of IL-10 was much higher than in the model group during the whole implantation time (P<0.01). It was suggested Bangderun may favor a shift from Th1- to Th2-type immune response, therefore inhibiting the maternal immune rejection, inducing the immune tolerance and improving the fetal implantation.
Drugs, Chinese Herbal/*pharmacology ; Embryo Implantation, Delayed/*drug effects ; Embryo Implantation, Delayed/immunology ; Endometrium/*immunology ; Endometrium/metabolism ; Interferon-gamma/genetics ; Interferon-gamma/metabolism ; Interleukin-10/genetics ; Interleukin-10/metabolism ; NF-kappa B/genetics ; NF-kappa B/metabolism

OBJECTIVESTo study the role of histone modification in the regulation of IFN-gamma-activated gene using chromatin immunoprecipitation technique.

METHODSReal-time PCR was used to measure the mRNA level of interferon-gamma-inducible protein 10 (IP-10) in Hela cells. Chromatin immunoprecipitation combined with Real-time PCR was used to check the histone H4 acetylation level at IFN-stimulated response element (ISRE) locus of IP-10 gene.

RESULTSIP-10 was strongly activated by IFN-gamma. The histone H4 deacetylation happened at the ISRE locus when IP-10 was induced by IFN-gamma. The activation of IP-10 and the deacetylation of histone H4 at the ISRE site induced by IFN-gamma were inhibited or blocked by histone deacetylase (HDAC) inhibitor trichostatin A (TSA).

CONCLUSIONThe histone H4 deacetylation at the ISRE site is related with the activation of IP-10 by IFN-gamma.

Acetylation ; Chemokine CXCL10 ; metabolism ; Gene Expression Regulation ; HeLa Cells ; Histone Deacetylases ; genetics ; metabolism ; Histones ; genetics ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; RNA, Messenger ; genetics

BACKGROUNDAlthough the onset of anemia during infectious disease is commonly correlated with production of inflammatory cytokines, the mechanisms by which cytokines induce anemia are poorly defined. This study focused on the mechanism research.

METHODSDifferent types of mice were infected perorally with Toxoplasma gondii strain ME49. At the indicated times, samples from each mouse were harvested, processed, and analyzed individually. Blood samples were analyzed using a Coulter Counter and red blood cell (RBC) survival was measured by biotinylation. Levels of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and inducible protein 10 (IP-10) mRNA in liver tissue were measured by real-time polymerase chain reaction.

RESULTST. gondii-infected mice exhibited anemia due to a decrease in both erythropoiesis and survival time of RBC in the circulation (P < 0.02). In addition, infection-stimulated anemia was associated with fecal occult, supporting previous literature that hemorrhage is a consequence of T. gondii infection in mice. Infection-induced anemia was abolished in interferon gamma (IFN&gamma;) and IFN&gamma; receptor deficient mice (P < 0.05) but was still evident in mice lacking TNF-α, iNOS, phagocyte NADPH oxidase or IP-10 (P < 0.02). Neither signal transducer and activator of transcription 1 (STAT1) deficient mice nor 129S6 controls exhibited decreased erythropoiesis, but rather suffered from an anemia resulting solely from increased loss of circulating RBC.

CONCLUSIONSInfection-stimulated decrease in erythropoiesis and losses of RBC have distinct mechanistic bases. These results show that during T. gondii infection, IFN&gamma; is responsible for an anemia that results from both a decrease in erythropoiesis and a STAT1 independent loss of circulating RBC.

Anemia ; genetics ; metabolism ; Animals ; Erythrocytes ; pathology ; Interferon-gamma ; metabolism ; Male ; Mice ; Mice, Knockout ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Receptors, Interferon ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Toxoplasma ; pathogenicity ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

This study investigated the mechanism of baicalin on lipopolysaccharide(LPS)/interferon γ(IFN-γ)-induced inflammatory microglia based on the triggering receptor expressed on myeloid cells 2(TREM2)/Toll-like receptor 4(TLR4)/nuclear factor kappaB(NF-κB) pathway. Specifically, LPS and IFN-γ were used to induce inflammation in mouse microglia BV2 cells. Then the normal group, model group, low-dose(5 μmol·L~(-1)) baicalin group, medium-dose(10 μmol·L~(-1)) baicalin group, high-dose(20 μmol·L~(-1)) baicalin group, and minocycline(10 μmol·L~(-1)) group were designed. Cell viability was detected by CCK-8 assay and cell morphology was observed under bright field. The expression of interleukin-1β(IL-1β), interleukin-4(IL-4), inducible nitric oxide synthase(iNOS), interleukin-6(IL-6), interleukin-10(IL-10), and arginase-1(Arg-1) mRNA was detected by real-time quantitative PCR, the protein expression of tumor necrosis factor-α(TNF-α), IL-1β, TREM2, TLR4, inhibitor kappaB-alpha(IκBα), p-IκBα, NF-κB p65 and p-NF-κB p65 by Western blot, and transfer of NF-κB p65 from cytoplasm to nucleus by cellular immunofluorescence. Compared with the normal group, most of the BV2 cells in the model group tended to demonstrate the pro-inflammatory M1 amoeba morphology, and the model group showed significant increase in the mRNA levels of IL-1β, IL-6, and iNOS, decrease in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), rise of the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.01), reduction in TREM2 protein expression, and increase in the expression of NF-κB p65 in nucleus. Compared with the model group, baicalin groups and minocycline group showed the recovery of BV2 cell morphology, significant decrease in the mRNA levels of IL-1β, IL-6 and iNOS, increase in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), reduction in the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.05), rise of TREM2 protein expression, and decrease in the expression of NF-κB p65 in nucleus. In summary, these results suggest that baicalin can regulate the imbalance between TREM2 and TLR4 of microglia and inhibit the activation of downstream NF-κB, thus promoting the polarization of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype.
Animals ; Flavonoids ; Inflammation/genetics* ; Interferon-gamma ; Lipopolysaccharides/adverse effects* ; Mice ; NF-kappa B/metabolism* ; Toll-Like Receptor 4/metabolism*

OBJECTIVETo elucidate the roles of JAK/STATs signal pathway on anti-proliferative effects induced by IFN-alpha in MHCC97.

METHODSAn IRF9 expression vector was transfected into MHCC97 with Dosper. The expression of IRF9, cycle regulating proteins and the forming of ISGF3 complex were detected using Western blot and EMSA, respectively. Cell proliferation and distribution were monitored using MTT and flow cytometry.

RESULTSHigh expression of IRF9 restored the anti-proliferative response of MHCC97 on IFN-alpha treatment and delayed the cell transition from S phase to G2 phase induced by IFN-alpha.

CONCLUSIONThe integrity and functions of JAK/STATs signal pathway played an important role in mediating the anti-proliferative effects of IFN-alpha in MHCC97.

Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Interferon-Stimulated Gene Factor 3, gamma Subunit ; genetics ; Interferon-alpha ; metabolism ; pharmacology ; Janus Kinases ; genetics ; physiology ; Liver Neoplasms ; genetics ; metabolism ; pathology ; STAT Transcription Factors ; genetics ; physiology ; Signal Transduction ; Transfection

In order to study PBD-2 and PoIFNgamma, the chimeric gene PBD-2-PoIFNgamma was synthesized by overlap extension PCR, and amplified PoIFNgamma on the basis of this sequence, then cloned into yeast expression vector pPICZalphaA separately to get the recombinant plasmid pPICZalphaA-PBD-2-PoINFgamma and pPICZalphaA-PoINFgamma. The recombinant plasmid was digested by Sac I and introduced into Pichia pastoris X33 cells by electroporation. Positive clones were screened and cultivated in BMMY medium containing 0.5% methanol for 72 h. SDS-PAGE and Western blotting analysis showed that the screened recombinant could secrete PBD-2-PoINFgamma and PoINFgamma separately. The activity of fusion protein was not detected by cytopathic effect inhibition assay and agar diffusion assay, but detected obvious antiviral activity of PoINFgamma. The helix and random coil contents was showed vary greatly between PoIFNgamma and PBD-2-PoLNFgamma by circular dichroism analysis. It was speculated that the fusion protein was not correctly folded and may affect the activity of PBD-2-PoINFgamma.
Animals ; Genetic Vectors ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Swine ; beta-Defensins ; biosynthesis ; genetics

In order to characterize the biological activity of fox (Vulpes vulpes) interferon gamma(VuIFN-gamma), We have isolated the cDNA encoding arctic fox (Alopex lagopus) VuIFN-gamma. This cDNA encodes a 23 amino acid signal peptide and a 144 amino acid mature protein, which shares 99.8% or 99.4% for nucleotide identity with silver fox and canine, respectively, and 100% for amino acid identity. Expression of recombinant mature arctic fox interferon gamma (mVuIFN-gamma) in bacterial system was confirmed by SDS-PAGE and Western blotting analysis. Recombinant VuIFN-gamma showed higher antiviral activity against vesicular stomatitis virus in cultured Vero and MDCK by inhibiting virus induced cytopathic effect, In view of the immunomodulatory and antiviral activities of VuIFN-gamma, it may provide a basis for further research on antiviral therapy of recombinant VuIFN-gamma in economic animal practice.
Animals ; Antiviral Agents ; pharmacology ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Foxes ; genetics ; Interferon-gamma ; genetics ; pharmacology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; genetics ; pharmacology

OBJECTIVETo elucidate the effects of interferon-gamma (IFN-gamma) on the transforming growth factor beta (TGF-beta)/Smad pathway in keloid-derived fibroblasts (KFb), and to investigate the underlying mechanism in the treatment of pathologic scar with IFN-gamma.

METHODSKeloid tissue of 3 patients were obtained, and then KFb were separated and cultured in vitro. KFb from passages 3 to 5 were used for the study. (1) KFb were divided into control group (incubated with serum-free DMEM), TGF-beta(1) group (treated with 10 ng/mL TGF-beta(1)), IFN-gamma group (treated with 100 ng/mL IFN-gamma), and TGF-beta(1)+IFN-gamma group (incubated with 10 ng/mL TGF-beta(1) combined with 100 ng/mL IFN-gamma). The expression level of mRNA and protein of connective tissue growth factor (CTGF), alpha smooth muscle actin (alpha-SMA) protein and expression of alpha-SMA positive KFb were detected by real-time fluorescent quantitation RT-PCR (FQ-RT-PCR), Western blot and immunofluorescence cytochemical staining. (2) Another sample of KFb was obtained and treated with 10 ng/mL IFN-gamma. The expression level of Smad 3 and Smad 7 protein was detected by Western blot before and 1, 2, 4, 6, 8 h post stimulation (PSH). The expression level of Smad 3 and Smad 7 mRNA was assessed by FQ-RT-PCR before stimulation and 30 mins post stimulation and at PSH, 1, 2, 4, 6, 8. (3) Another sample of KFb was obtained and divided into 1, 10 and 100 ng/mL IFN-gamma groups based on the concentration of IFN-gamma, treated for 4 hours; KFb without IFN-gamma treatment was set up as control group. The expression levels of the protein and mRNA of Smad 3 and Smad 7 were measured by FQ-RT-PCR and Western blot.

RESULTS(1) The level of mRNA and protein of CTGF in IFN-gamma group (0.017 +/- 0.009 and 1.198 +/- 0.004) was respectively lower than that in control group (0.024 +/- 0.013 and 1.229 +/- 0.011, P < 0.05). The level of mRNA and protein of CTGF in TGF-beta(1)+IFN-gamma group (0.634 +/- 0.138 and 1.204 +/- 0.010) was respectively lower than that in TGF-beta(1) group (1.331 +/- 0.298 and 1.727 +/- 0.004, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (0.922 +/- 0.059) and the expression level of alpha-SMA protein (0.3051 +/- 0.0031) in IFN-gamma group decreased significantly than those in control group (1.055 +/- 0.005 and 0.4513 +/- 0.0094, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (1.129 +/- 0.004) and the expression level of alpha-SMA protein (0.6734 +/- 0.0098) in TGF-beta(1)+IFN-gamma group decreased significantly than those in TGF-beta(1) group (1.270 +/- 0.005 and 1.3842 +/- 0.0024, P < 0.01). (2) The expression level of Smad 3 mRNA and protein at the first time point after IFN-gamma treatment increased temporarily then decreased gradually, and mRNA expression level reached the nadir at PSH 4, it rose gradually later, though it was still lower at PSH 8 than that before treatment (P < 0.01); protein expression level at PSH 8 was significantly lower than that before treatment (P < 0.01). The expression level of Smad 7 mRNA and protein increased gradually to the maximum at PSH 2 and 4 respectively, then decreased but was still higher at PSH 8 than that before treatment (P < 0.05). (3) Compared with those in control group, the expression levels of Smad 3 mRNA and protein in 1, 10 and 100 ng/mL IFN-gamma group were significantly lower, the expression levels of Smad 7 mRNA and protein were significantly higher (P < 0.05 or P < 0.01). The higher concentration of IFN-gamma, the more significant differences were observed.

CONCLUSIONSIFN-gamma can down-regulate the expression of Smad 3 while up-regulate the expression of Smad 7 in a time- and dose-dependent manner, and reduce the expression level of CTGF and alpha-SMA in the basic state or induced by TGF-beta(1), which shows a significant inhibitory effect on the TGF-beta/Smad signal pathway. This may be an important mechanism in the treatment of pathologic scar by IFN-gamma.

Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Keloid ; metabolism ; RNA, Messenger ; genetics ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta1 ; metabolism