The total RNA was extracted from peripheral blood mononuclear cells (PBMC) which was isolated from Meishan porcine and induced with concanavaline A (ConA), then the porcine interferon gamma gene (PoIFNgamma, 501bp) was amplified by RT-PCR. The result of sequencing demonstrated that the amplified PoIFNgamma had 100% nucleotide homology with the other porcine IFNgamma sequence published on GenBank. The objective gene (PoIFNgamma) was inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNgamma. And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37 degrees C for 24h on kanamycin resistance plate and selected for smaller colonies. Then, the extracted recombinant plasmid was named pAd-Sh-PoIFNgamma, which was confirmed by Pac I digestion, and transformed into XL10-Glod(r) for copious preparation. pAd-Sh-PoIFNgamma linearized with Pac I was co-transfected with liposome into 293 package cell-line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome (deleted with E1 and E3 genes) carrying PoIFNgamma was successfully packaged into intact virion. The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR. The antiviral activity of PoIFNgamma was tested by CPE50 method. The results showed that the PoIFNgamma expressed by adenovirus had high antiviral activity, which was 1.3 x 10(6) U/mL against VSV in MDBK cells. The results demonstrated that the recombinant adenovirus carrying PoIFNgamma could be stably passaged.
Adenoviridae ; genetics ; Animals ; Antiviral Agents ; pharmacology ; Interferon-gamma ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; pharmacology ; Swine

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×10⁷ U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.
Swine ; Animals ; Cricetinae ; Interferon-gamma/pharmacology* ; Cricetulus ; CHO Cells ; Sincalide ; Recombinant Proteins/pharmacology* ; Antiviral Agents/pharmacology*

BACKGROUNDHaptoglobin (Hp) is one of the acute-phase proteins. Recent studies have demonstrated that Hp exerts immunoregulatory and anti-inflammatory actions and may be one of the inhibitory factors of immune reactions in the skin. In this study we investigated the regulation of Hp expression in a human keratinocyte cell line HaCaT by various cytokines and glucocorticoid.

METHODSHaCaT cells were cultured with IL-6 (50 ng/ml), TNF-alpha (20 ng/ml), IFN-gamma (20 ng/ml) or IL-4 (20 ng/ml) with or without 1 micromol/L dexamethasone in 6-well plates for 12, 24 and 48 hours. Both the cells and the supernatants were collected to detect the changes of Hp expression by reverse-transcription PCR, ELISA and immunohistochemistry.

RESULTSThe results showed that Hp expression were elevated at both the mRNA and protein level by the combination of IL-6, TNF-alpha or IL-4 with dexamethasone, whereas the three cytokines alone did not upregulate the Hp expression. IFN-gamma showed no effect on the Hp expression in HaCaT cells.

CONCLUSIONSThese findings suggest that different inflammatory cytokines as well as glucocorticoid may be involved in the regulation of Hp expression in keratinocytes, and this may be one of the negative feedback mechanisms in inflammatory skin diseases.

Cell Line ; Dexamethasone ; pharmacology ; Glucocorticoids ; pharmacology ; Haptoglobins ; analysis ; Humans ; Interferon-gamma ; pharmacology ; Interleukin-4 ; pharmacology ; Interleukin-6 ; pharmacology ; Keratinocytes ; chemistry ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo observe the effect of Shenghua Decoction in reversing IFN-gamma inhibition induced by methotrexate (MTX).

METHODSPregnant Balb/C mice were randomly divided into the negative control group, Shenghua Decoction group, the MTX control group, and the MTX combined with Shenghua Decoction group. Pregnant mice in the 4 groups were respectively treated with normal saline (NS), Shenghua Decoction, MTX, and MTX combined with Shenghua Decoction for 7 successive days. Then mice were sacrificed 1 h after the last administration. Living fetus and dead fetus were counted to assess meto-maternal rejection. The percentages of interferon-gamma+(IFN-gamma+) and CD3+CD+IFN-gamma+ cells in spleen lymphocytes of pregnant mice were double stained for cytometric analysis. At the same time IFN-gamma message ribonucleic acid (mRNA) in the spleen mononuclear cells was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSCompared with the negative control group, the number of dead fetus increased significantly after the administration of Shenghua Decoction (P > 0.05). The percentages of IFN-gamma+ and CD3+CD4+IFN-gamma+ in spleen mononuclear cells were elevated by 28.81% and 35.61% respectively (both P < 0.05). IFN-gamma mRNA expression increased (P < 0.05). After the administration of MTX, the number of dead fetus increased significantly. The percentages of IFN-gamma+ and CD3+CD4+IFN-gamma+ in spleen mononuclear cells were reduced by 24.23% and 26.77% respectively compared with the negative control group. Besides, the expression of IFN-gamma mRNA decreased obviously (P < 0.05). However, after the administration of MTX in combination of Shenghua Decoction, the number of dead fetus was further added. Moreover, the percentages of IFN-gamma+ and CD3+CD4+IFN-gamma+ cells obviously increased. The increment was 64.96% and 71.38% respectively when compared with the MTX group, and the expression of IFN-gamma mRNA was obviously up-regulated (P < 0.05).

CONCLUSIONShenghua Decoction showed reversing effect on MTX induced IFN-gamma inhibition by promoting the differentiation of IFN-gamma+Th1 subpopulation and up-regulating IFN-gamma expression, thus promoting the feto-maternal immunological rejection.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Immunosuppression ; Interferon-gamma ; immunology ; Methotrexate ; pharmacology ; Mice ; Mice, Inbred BALB C ; Pregnancy

This study was aimed to explore the killing effect of PBMNC induced by IL-23 alone or combined with IL-2 on K562 cells and its mechanism. The PBMNC were induced in vitro by IL-23 (50 ng/ml) alone or IL-23 combined with IL-2 (100 U/ml) for 72 h, and then were co-cultured with leukemia cell line K562. The CCK-8 method was used to detect the effect of PBMNC induced at different times on K562 cells, the ELISA was performed for detecting IFN-&gamma; level in culture supernatant, and the perforin and granzymes B were detected by RQ-PCR. The results showed that the killing effect of PBMNC induced by IL-23 alone or IL-23 combined with IL-2 on K562 cells was observed, and obviously enhanced with prolonging of time, moreover, there was statistical difference among different time points (P < 0.05). The IFN-&gamma; level in supernatant of PBMNC cultured with cytokines significantly increased, and the IFN-&gamma; levels in group of IL-23 combined with IL-2 were higher than that in other groups (P < 0.05). The mRNA expressions level of perforin and granzymes B of the expanded PBMNC in groups cultured with cytokines were higher than that in control group (P < 0.05), and the mRNA expressions of perforin and granzymes B in group of IL-23 combined with IL-2 were significantly higher than that in others (P < 0.05). It is concluded that IL-23 can promote the killing effect of PBMNC on K562 cells. The combination of IL-2 with IL-23 displays synergic effect and a time-dependent manner. IL-23 also enhances the expression of IFN-&gamma;, perforin and granzyme B in PBMNC. Its combination with IL-2 displays synergistic effect, suggesting that the anti-leukemic activity of IL-23 may be realized through inducing PBMNC to express IFN-&gamma;, perforin and granzyme B.
Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-2 ; pharmacology ; Interleukin-23 ; pharmacology ; K562 Cells ; Monocytes ; drug effects ; metabolism ; Perforin ; metabolism

This study was objective to explore the effect of IFN-&gamma; on immunosuppressive capability of mesenchymal stem cells (MSC) derived from umbilical cord. The immunomodulating capability of MSC was changed by stimulating cell surface receptors like Toll-like receptors (TLR). The inhibition of T-lymphocyte proliferation by MSC was tested via cell co-cultures. Further RT-PCR and ELISA were performed to examine the expression changes in gene and protein level. The results showed that the IFN-&gamma; could promote the immunosuppressive effect of umbilical cord derived MSC. IFN-&gamma;-stimulated MSC could suppress the proliferation of T cells more effectively. IFN-&gamma; stimulation up-regulated the expression of immunosuppressive genes like IDO1, COX2, HLA-G, and soluble suppressive proteins such as HLA-G, KYN, IL10, PGE2 of MSC. And the immuno suppression capability of IFN-&gamma;-stimulated MSC was 2-7 folds higher than control in MSC and lymphocyte co-culture tests. It is concluded that IFN-&gamma; can effectively enhance the immunosuppressive capability of MSC.
Cells, Cultured ; Humans ; Immunosuppression ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology ; Umbilical Cord ; cytology

The objective of study was to investigate the relationship between expressions of CIITA and MHC molecules in five human cell lines. The expressions of MHC molecules and CIITA protein were detected by Western blot, immunohistochemistry and flow cytometry. The expression of CIITA gene was measured by RT-PCR. The results indicated that the expression of MHC-II molecules in 5 human cell lines was consistent with expression of CIITA. The cell lines constitutively expressed CIITA also expressed MHC-II molecules, the expression of MHC-II molecules in cell lines expressed CIITA after induction with IFN-gamma also recovered; the cell lines unexpressed CIITA after induction with IFN-gamma did not respond to IFN-gamma-promoting expression of MHC-II molecules. It is concluded that some cell lines cannot express MHC-II molecules which may be related with deficiency of CIITA expression. It suggest that CIITA participates in regulation of MHC-II molecule expression, which may plays a certain role in escape from carcinogenesis under surveillance of immune system.
Cell Line ; Genes, MHC Class II ; Humans ; Interferon-gamma ; pharmacology ; Nuclear Proteins ; metabolism ; Trans-Activators ; metabolism ; Tumor Cells, Cultured

This study was purposed to investigate the effects of interferon (IFN)-&gamma; on expression of adhesion molecules in mesenchymal stromal cells derived from human umbilical cord tissue (UC-MSC). The UC-MSC were isolated from human umbilical cord by tissue culture. The expressions of specific markers on UC-MSC were detected by flow cytometry in the physiological condition. The adipogenic and osteogenic induction of UC-MSC was detected by alizarin and Oil red O staining. UC-MSC were exposed to IFN-&gamma; (100, 1 000, 10 000 U/ml) for 24 h, the expressions of CD54, CD58, CD44, CD49d, CD62p, CD62L, CD102 and CD106 on cell surface were detected using flow cytometry. The results showed that in physiological condition, UC-MSC extremely low expressed CD102, CD106, CD62P, CD62L, while the expression of CD54 was relatively high (41.58 ± 0.83)%. When stimulated by IFN-&gamma;, the expression of CD102, CD106, CD62P, CD62L increased slightly, but still low (< 5%), while CD54 and CD58 upregulated concentration-dependently up to (59.66 ± 1.36)% and (43.96 ± 0.62)% respectively. The expression of CD49d upregulated to (51.33 ± 0.74)% when UC-MSC exposed to IFN-&gamma; 100 U/ml. CD44 increased to (73.22 ± 1.93)% when UC-MSC exposed to IFN-&gamma; 1 000 U/ml. It is concluded that IFN-&gamma; can elevate significantly the expression of CD54, CD49d, CD44 and CD58, but has no significant effect on CD102, CD106, CD62P and CD62L expression on the surface of UC-MSC.
Cell Adhesion Molecules ; metabolism ; Cells, Cultured ; Humans ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Umbilical Cord ; cytology

OBJECTIVETo observe the inhibitory effect of a eukaryotic expression vector expressing human IFN-gamma (pcDNA3.1- IFN-gamma) on HBV replication in hepG2.2.15 cells.

METHODSThe eukaryotic expression vector expressing human IFN-gamma was constructed using PCR and gene recombination technique. hepG2.2.15 cells were transfected with pcDNA3.1-IFN-gamma and the culture supernatant was collected to determine the expression of IFN-gamma protein by ELISA. The HBV DNA copies and the concentration of HBeAg and HBsAg were measured by fluorescence real-time PCR and ELISA kit, respectively.

RESULTSCompared with that of negative control and blank 2.2.15 cells, the concentration of HBeAg in the supernatant of 2.2.15 cells transfected with pcDNA3.1- IFN-gamma were decreased by 49%, and HBsAg concentration was lowered by 35% and 33%, respectively. A significant decrease of HBV DNA copies was observed in pcDNA3.1- IFN-gamma-transfected cells in comparison with the two control cells. No significant differences were noted in all the results between the two control groups.

CONCLUSIONWe have successfully constructed the eukaryotic expression vector expressing human IFN-gamma, which provides a basis for anti-HBV gene therapy using human IFN-gamma.

Genetic Vectors ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; Humans ; Interferon-gamma ; genetics ; pharmacology ; Transfection ; Virus Replication ; drug effects

OBJECTIVE: To observe the effect of interferon-gamma on the expression of vascular endothelial growth factor C on Hep-2 laryngeal carcinoma cell lines. METHOD: Hep-2 cell was exposed to interferon-gamma at a concentration (10(3), 10(4), 10(5), 10(6), 10(7) U/L) for 0, 12, 24, 36, 48, 60, 72 h hours. MTT colorimetric assay was used to evaluate the effect of interferon-gamma on Hep-2 cell proliferation after incubation with interferon-gamma. Hep-2 cell was harvested to be detected VEGF-C mRNA levels by realtime polymerase chain reaction (Realtime-PCR) with interferon-gamma (10(6) U/L) for 0, 2, 6, 12, 24, 36, 48, 60, 72 h hours. The VEGF-C protein level in supernatants was determined by enzyme-linked immunosorbent assay (ELISA) and in cytoplasma by immunocytochemical staining and Western blot analysis with the same drug treatment as Realtime-PCR. RESULT: Hep-2 cell was significantly inhibited by IFN-gamma at high concentration. Expression of VEGF-C mRNA and protein levels of Hep-2 cell at 10(6) U/L concentrations of interferon-gamma were lower than that of normal control (P < 0.05) at 48 h. CONCLUSION IFN-gamma can inhibit the expression of VEGF-C in Hep-2 cell with the level of mRNA and protein.
Cell Line, Tumor ; Humans ; Interferon-gamma ; pharmacology ; Laryngeal Neoplasms ; metabolism ; pathology ; Vascular Endothelial Growth Factor C ; metabolism