Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-γ

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-γ, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-γat 0.1 to 5μg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5μg/L, IFN-γalso suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-γshowed no additive effect, sequential treatment first with lamivudine and then IFN-γwas found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2μmol/L lamivudine for two days, followed by 1μg/L IFN-γfor another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2μmol/L lamivudine for two days, followed by 5μg/L IFN-γfor six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-γand lamivudine, especially in IFN-αnon-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

Anti Cervix Cancer Activity of Co-immobilized Tumor Necrosis Factor-α and Interferon-γ

Tumor necrosis factor α （TNF-α） and interferon-γ （IFN-γ） are cytokines with strong antitumor activities. They were reacted with a photoactive arylazide-4-azidobenzoic acid, resulting in photoactive TNF-α and IFN-γ. The infrared （IR） spectra of these products showed the characteristic absorption of an azido group at 2127 cm^-1. By photo-immobilization, this modified TNF-α and IFN-γ were immobilized on polystyrene membranes for cell culture to prepare biomaterials. The micro-morphology of photoactive cytokines was observed with a scanning electron microscope （SEM）. The inhibitory effect on growth of Hela cells and inducing apoptosis activity of these two cytokines were analyzed by growth curve, transmission electron microscope （TEM） and fluorescence active cell sorter （FACS）. The results showed that co-immobilization of IFN-γ and TNF-α had significant inhibitory effect on growth of Hela cells, inhibitory rate up to 82%, and IFN-γ had obviously synergistic action.

Clinical Value of Vascular Endothelial Growth Factor Combined with Interferon-γ in Diagnosing Malignant Pleural Effusion and Tuberculous Pleural Effusion

In order to investigate the clinical value of vascular endothelial growth factor （VEGF） combined with interferon-γ （IFN-γ） in diagnosing malignant pleural effusion and tuberculous pleural effusion, 42 cases of malignant pleural effusion and 45 cases of tuberculous pleural effusion in Tongji Hospital, from March 2004 to May 2005, were included, The carcinoembryonic antigen （CEA）, VEGF and IFN-γ levels of pleural effusion were detected by using ELISA, and adenosine deaminase （ADA） activity was determined by using enzyme kinetic analytical method. The sensitivity, specificity, accuracy and area under the curve （AUCR^ROC） of CEA and VEGF, VEGF/IFN-γ ratio, ADA and IFN-γ were measured by receiver operating characteristic curve （ROC）, The results showed that CEA, VEGF levels and VEGF/IFN-γ ratio were significantly higher and the ADA and IFN-γ levels were significantly lower in malignant group than those in tuberculous group （P〈0,01）, The sensitivity, specificity, accuracy and AUCR^ROC of VEGF/IFN-γ ratio （88,7%, 99,8%, 94,4%, 0.96 respectively） were higher than those of CEA （67.8%, 96.1%, 82,4%, 0.78 respectively） and VEGF （81,5%, 84,3%, 82.9%, 0.79 respectively）. The sensitivity, specificity, accuracy and AUCR^ROC of IFN-γ （85.7%, 96,4%, 90.9%, 0.94 respectively） were higher than those of ADA （80,2%, 87,6%, 83.8%, 0,81 respectively）. It was concluded that VEGF/IFN-γ ratio and IFN-γ could be used as valuable parameters for the differential diagnosis of malignant pleural effusion and tuberculous pleural effusion.

Failure of interferon-γ pre-treated mesenchymal stem cell treatment in a patient with crohn's disease

Mesenchymal stem cells(MSC) are cells of stromal origin which exhibit unlimited self-renewal capacity and pluripotency in vitro.It has recently been observed that MSC may also exert a profound immunosuppressive and anti-inflammatory effect both in vitro and in vivo with consequent potential use in autoimmune disorders.We present the case of a patient suffering from childhood-onset, multidrug resistant and steroiddependent Crohn's disease who underwent systemic infusions of MSC, which led to a temporary reduction in CCR4, CCR7 and CXCR4 expression by T-cells, and a temporary decrease in switched memory B-cells, In addition, following MSC infusion, lower doses of steroids were needed to inhibit proliferation of the patient's peripheral blood mononuclear cells.Despite these changes, no significant clinical benefit was observed, and the patient required rescue therapy with infliximab and subsequent autologous hematopoietic stem cell transplantation.The results of biological and in vitro observations after MSC use and the clinical effects of infusion are discussed, and a brief description is provided of previous data on MSC-based therapy in autoimmune disorders.

Interferon-γ: Promising therapeutic target in atherosclerosis

Atherosclerosis is a chronic inflammatory disorder of the vasculature and is the primary cause of cardiovascular disease(CVD). CVD is currently the world's leading cause of death and the numbers are predicted to rise further because of a global increase in risk factors such as diabetes and obesity. Current therapies such as statins have had a major impact in reducing mortality from CVD. However, there is a marked residual CVD risk in patients on statin therapy. It is therefore important to understand the molecular basis of this disease in detail and to develop alternative novel therapeutics. Interferon-γ(IFN-γ) is a pro-inflammatory cytokine that is often regarded as a master regulator of atherosclerosis development. IFN-γ is able to influence several key steps during atherosclerosis development, including pro-inflammatory gene expression, the recruitment of monocytes from the blood to the activated arterial endothelium and plaque stability. This central role of IFN-γ makes it a promising therapeutic target. The purpose of this editorial is to describe the key role IFN-γ plays during atherosclerosis development, as well as discuss potential strategies to target it therapeutically.

Interferon-γ inhibits ghrelin expression and secretion via a somatostatin-mediated mechanism

AIM- To investigate if and how the proinflammatory cytokine interferon γ （IFNγ） affects ghrelin expression in mice. METHODS： The plasma concentration of ghrelin, and gastric ghrelin and somatostatin expression, were ex- amined in wild-type mice and mice infected with Helico- bacter pylori （H. pylorO. Furthermore, ghrelin expression was examined in two achlorhydric mouse models with varying degrees of gastritis due to bacterial overgrowth. To study the effect of IFNγ, alone, mice were given a subcutaneous infusion of IFNγ, for 7 d. Finally, the influ- ence of IFNγ, and somatostatin on the ghrelin promoter was characterized. RESULTS： H. py/ori infection was associated with a 50% reduction in ghrelin expression and plasma concentration. Suppression of ghrelin expression was in- versely correlated with gastric inflammation in achlorh- dyric mouse models. Subcutaneous infusion of IFNγ, suppressed fundic ghrelin mRNA expression and plasma ghrelin concentrations. Finally, we showed that the ghrelin promoter operates under the control of soma- tostatin but not under that of IFNγ. CONCLUSION： Gastric infection and inflammation is associated with increased IFNγ, expression and reduced ghrelin expression. IFNγ, does not directly control ghre- lin expression but inhibits it indirectly via somatostatin.

Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment

AIM： To evaluate anti-hepatitis B virus （HBV） activity and cytotoxicity of interferon-α, （IFN-3,） and tumor ne- crosis factor-α （TNF-α） following lamivudine treatment of HepG2.2.15 cells. METHODS： HepG2.2.15 cells were treated with 2 pmol/L lamivudine for 16 d （lamivudine group）, cultured for 10 d, followed by 5 ng/ml TNF-α and 1000 U/mL IFN-γ, for 6 d （cytokine group）, or treated with 2 ~tmol/L lami- vudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ, for 6 d （sequential group）, or cultured without additions for 16 d （control group）. Intracellular DNA was extracted from 3 ×10^ HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circu- lar DNA that may have remained. Both HBV covalently closed circular DNA （cccDNA） and HBV DNA were exam- ined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg） were quantified with enzyme-linkedimmunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS： Compared to lamivudine alone （22.63%±0.12%）, both sequential （51.50% ± 0.17%, P = 0.034） and cytokine treatment （49.66% ± 0.06%, P = 0.041） showed a stronger inhibition of HBV cccDNA; the dif- ference between the.sequential and cytokine groups was not statistically significant （51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88）. The sequential group showed less inhibition of HBV DNA replication than the lamivudine group （67.47% ±0.02% vs 82.48% ± 0.05%, P = 0.014）; the difference between the sequen- tial and cytokine groups was not statistically significant （67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071）. The levels of HBsAg and HBeAg were significantly de- creased in the sequential treatment group compared to the other groups [HBsAg： 3.48 ± 0.04 （control）, 3.09 ± 0.08 （lamivudine）, 2.55± 0.13 （cytokine）, 2.32 ± 0.08 （sequential）, P = 0.042 for each between-group comparison; HBeAg 3.48 ± 0.01 （control）, 3.08 ± 0.08 （lamivudine）, 2.57 ± 0.15 （cytokine）, 2.34 ± 0.12 （se- quential）, P = 0.048 for each between-group compari- son]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells （58.03% ± 8.03% vs 100%, P = 0.000）. Lamivudine pretreat- ment significantly reduced IFN-γ, ± TNF-αmediated toxicity of HepG2.2.15 cells [85.82% =1= 5.43% （sequen- tial） vs 58.03% ± 8.03% （cytokine）, P = 0.002]. CONCLUSION： Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-y and TNF-α by decreasing the viral load.

Interferon-γ acts as a regulator in the trade-off between phagocytosis and production performance in dwarf chickens

Background: Interferon-γ(IFN-γ) is critical for innate and adaptive immunity against viral and bacterial infections. IFN-γ reportedly affects the phagocytic ability of monocytes and macrophages as well as regulates pituitary function in humans and mice. The present study analyzed the impact of IFN-γ on monocyte and macrophage phagocytosis, production performance, and pituitary function in vivo and in vitro(in dwarf chickens). IFN-γ was injected into dwarf chickens through a vein, and then, the laying rate, average egg weight, and levels of follicle-stimulating hormone(FSH) and IFN-γ were measured in treatment and control groups. For the in vitro experiment, the pituitary tissues were supplemented with IFN-γ, and the m RNA expression levels of follicle-stimulating hormone beta subunit(FSH-β), interferon gamma receptor 1(IFNGR1),and interferon gamma receptor 2(IFNGR2) in the pituitary were assessed.Results: Monocyte and macrophage phagocytosis product(PP) was decreased by IFN-γ treatment in a dose-dependent manner in vitro. In the in vivo experiment, the level of IFN-γ in the treatment group was higher than that in the control group at 7 d(P < 0.05), 14 d(P < 0.01), and 21 d(P < 0.01) post-injection.Compared with the control group, monocyte and macrophage PP was lower in the treatment group after injection(P < 0.01). The laying rate was higher in the treatment group than in the control group at 2 and3 wk post-injection(P < 0.05). There was a significant difference between the treatment and control groups in the levels of FSH at 1, 3, 7, and 14 d post-injection(P < 0.01). In the in vitro experiment, increased m RNA expression levels of FSH-β, IFNGR1, and IFNGR2 were observed in the treatment group after stimulation with100 U/m L IFN-γ for 24 h compared to those in the control group(P < 0.05).Conclusions: IFN-γ inhibited the phagocytosis of monocytes and macrophages; up-regulated the m RNA expression levels of the FSH-β, IFNGR1, and IFNGR2; enhanced the secretion of FSH; and improved the laying rate. IFN-γ might be an important regulator in the trade-off between the immune effect and production performance in dwarf chickens.

Interferon-γ mRNA attenuates its own translation by activating PKR： A molecular basis for the therapeutic effect of interferon-β in multiple sclerosis

PKR, the interferon （IFN）-inducible protein kinase activated by double-stranded RNA, inhibits translation by phosphorylating the initiation factor eIF2α chain. Uniquely, human IFN-γ mRNA uses local activation of PKR in the cell to control its own translation yield. IFN-γ mRNA activates PKR through a structure in its 5＇- region harboring a pseudoknot which is critical for PKR activation. Mutations that impair pseudoknot stability reduce the ability of IFN-γ mRNA to activate PKR and strongly increase its translation efficiency. The cis-acting RNA element in IFN-γ mRNA functions as a biological sensor of intracellular PKR levels. During an immune response, as IFN-γ and other inflammatory cytokines build up in the cell＇s microenvironment, they act to induce higher levels of PKR in the cell, resulting in a more extensive activation of PKR by IFN-γ mRNA. With the resulting phosphorylation of eIF2α, a negative feedback loop is created and the production of IFN-γ is progressively attenuated. We propose that the therapeutic effect of IFN-β in multiple sclerosis may rest, at least in part, on its exquisite ability to induce high levels of PKR in the cell and thereby to limit IFN-γ mRNA translation through this negative feedback loop, blocking the excessive IFN-γ gene expression that precedes clinical attacks.

EXTRACTION OF INTERFERON-γ USING PEG/K_3PO_4 AQUEOUS TWO-PHASE SYSTEM

The aqueous two-phase system of PEG-4000/K3PO4 is selected to extract interferon-γ（γ-IFN）from the broth of homogenized E.coil cells,which were recombined genetically and werefermented in high expressivity.The effects of pH value and the concentrations of PEG and phos-phate on the partition behavious of γ-IFN and contaminating proteins have been investigated.Theresults show that the pH value is the most influential factor effecting the extraction yield of γ-IFN.Under a suitable condition,an effective separation with high yield of γ-IFN can be achieved.

Myricetin inhibits interferon-γ-induced programmed death ligand-1 and indoleamine 2,3-dioxygenase 1 expression in lung cancer cells

OBJECTIVE Programmed death ligand-1(PD-L1)and indoleamine 2,3-dioxygenase 1(IDO1)are immune checkpoints which can be induced by interferon-γ(IFN-γ)in the tumor microenvironment,leading to immune escape of tumors.Myricetin(MY)is a flavonoid distributed in many edible and medicinal plants.The aim of this study is to clarify the effect and the mechanism of MY on inhibiting IFN-γ-induced PD-L1 and IDO1 in lung cancer cells.METHODS Expressions of PD-L1 and major histocompatibility complex-I(MHC-I)were evaluated by flow cytometry and Western blotting,and the expression of IDO1 was measured by Western blotting.qRT-PCR was used to detect their mRNA levels.The function of T cells was evaluated using a co-culture system consist of lung cancer cells and the Jurkat-PD-1 T cell line that overexpressing PD-1.Molecular docking analysis,Western blotting and immunofluorescence were used for mechanism study.RESULTS MY potently inhibited IFN-γ-induced PD-L1 and IDO1 expression in human lung cancer cells,while didn't show obvious effect on the expression of MHC-I.In addition,MY restored the survival,proliferation,CD69 expression and interleukin-2(IL-2)secretion of Jurkat-PD-1 T cells suppressed by IFN-γ-treated lung cancer cells in the co-culture system.Mechanistically,IFN-γup-regulated PD-L1 and IDO1 at the transcriptional level through the JAK-STAT-IRF1 axis,which was targeted and inhibited by MY.CONCLUSION Our research revealed a new insight into the anti-tumor effects of MY which inhibited IFN-γ-induced PD-L1 and IDO1 expression,supporting the potential of MY in anti-tumor immunotherapy.

Construction of Fluorescence Sensing Platform on the Basis of Carbon Nitride Nanosheet for the Detection of Interferon-γ

Purpose: Interferon-γ (INF-γ) is a cytokine that participates in the immune reaction of the body. Its level of secretion can reflect the immune response condition after the body is infected by pathogens, which is a significant indication of clinically-related diseases. Therefore, it is of great significance in application to develop a fluorescence biosensor to inspect INF-γ with rapidness, high sensitivity and high practicability. Method: The fluorescence sensor is made on the basis of the two-dimensional nano-material namely Carbon Nitride Nanosheet (CNNS) and the Aptamer probe to identify INF-γ (Apt&reg;INF-γ). CNNS can quickly quench the Cy5 fluorescent dye modified on the Apt&reg;INF-γ probe due to the Photoinduced Electron Transfer (PET), but when the INF-γ exists, Apt&reg;INF-γ specifically identifies and combines it. The complex of Apt&reg;INF-γ and INF-γ is away from CNNS, which can effectively block the fluorescent signal of Apt?INF-γ being quenched by CNNS. Result: The sensitive detection of IFN-γ protein can be achieved through the application of CNNS/Apt&reg;INF-γ fluorescence sensing platform. In this method, the intensity of the fluorescent signal is positively correlated with the concentration of IFN-γ, of which the liner response range is 0.5 - 100 ng/mL and the limit of detection is 0.303 ng/mL. In addition, this fluorescence sensing platform has the advantages of high specificity, simple operation and low costs. It can inspect the content of IFN-γ in clinical serum samples without interference. The actual recovery rate of serum samples is 97.11% - 106.96%. Conclusion: Therefore, the CNNS/Apt&reg;INF-γ sensing platform is expected to be implemented in the actual clinical detection, also conducive to developing a universal fluorescence biosensor to inspect other target materials.

Development and validation of a prognostic indicator for thyroid carcinoma based on interferon-γ-related gene analysis

Background:Women are mostly affected by thyroid carcinoma(THCA),an endocrine system cancer.However,the biomarkers of interferon-gamma(IFN-γ)in THCA have not been identified,so this study aimed to investigate whether IFN-γ-related genes could predict the overall prognosis of THCA patients.Methods:Transcriptome-related expression data were obtained from The Cancer Genome Atlas database.Differential expression of IFN-γ-responsive genes(DE-IFN-γ)between THCA and normal samples was determined based on the“limma”package in R.The prognostic value of the model was determined by least absolute shrinkage and selection operator,univariate Cox,and multivariate Cox analyses,as well as Kaplan-Meier curves.A nomogram was created to predict the THCA survival probabilities by combining clinicopathological features and prognostic genetic features.High-risk and low-risk groups were examined THCA-related pathways using gene set enrichment analysis.Correlations between the two groups with different scores and the immune microenvironment and immunotherapy were also explored.Finally,we verified the expression levels using real-time PCR.Results:From 48 DE-IFN-γ,4 DE-IFN-γ(METTL7B,VAMP8,CFB,IFIT3)associated with good prognosis were selected by least absolute shrinkage and selection operator and Cox co-screening.Based on these four genes,THCA patients were divided into two groups,with the high-risk group having a poorer overall survival rate.The risk score,age,and staging were identified as independent prognostic factors.The low-scoring group had significantly enriched 13 signaling pathways,according to gene set enrichment analysis.Meanwhile,the two groups delineated according to the risk score differed in terms of the immune microenvironment and immune checkpoints.Finally,our real-time PCR results corroborated previous hypotheses.Conclusion:Researchers identified four DE-IFN-γbiomarker genes with promising prognostic value for THCA patients,which may help guide immunotherapy preference.Moreover,it may subsequently influence our THCA treatment decisions.

The Effects of Triptolide on HLA Antigens Expression of Corneal Epithelial Cells Induced by Interferon-γ in Vitro

Objective:To observe the effects of immunosuppressants triptolide(TL)and cyclosporineA(CSA)on HLA antigens expression induced by interferon-γ（INF-γ）in vitro.Method:By using an indirect immunofluorescent method and analysing with ACAS-570,the abnormal HLA antigen expression of cultured corneal epithelial cells was induced by INF-γ，After incubation with one of the immunosuppressants(CSA,TL) for72hrs.the amount of HLA-ABCand HLA-DRantigens was measured.Result:There was no significant difference(P>0.05)between the group with CAS and the positive control group without CAS.In contrast to CSA,TL dramatically inhibited INF-γinduced expression of HLA antigens of corneal epithelial cells(P<0.001),compared with the control group withoutTL.Conclusion:TL had direct inhibition on the expression of HLA-ABCand HLA-DRantige ns induced by INF-γin vitro,while CSAhad no obvious inhibition,Eye Science2000;16:34-37.

Assessing the causality of interferon-γ and its receptor 1/2 with systemic lupus erythematosus risk using genetic data

Background::The interferon-γ (IFN-γ) signaling pathway is activated in systemic lupus erythematosus (SLE). This study aimed to assess the causal association between IFN-γ, IFN-γ receptor 1 (IFN-γR1), and IFN-γR2 and SLE using a bidirectional Mendelian randomization (MR) design.Methods::Genetic instruments for exposure to IFN-γ, IFN-γR1, and IFN-γR2 were derived from a large genome-wide association study (GWAS) that included a sample size of 3301 participants. Instrumental variables for SLE were selected from another independent GWAS analysis comprising 5201 cases and 6099 controls with European ancestry. Bidirectional two-sample MR was performed using inverse variance weighting, MR-Egger regression, and weighted median methods. A series of sensitivity analyses were conducted to assess the robustness of the results.Results::The inverse variance weighting showed that IFN-γ had a positive causal association with the risk of SLE (odd ratio [OR] = 1.24, 95% confidence interval [CI]: 1.03–1.47, p = 0.018). IFN-γR2 levels were not associated with SLE risk after adjustment for multiple comparisons (OR= 0.85, 95% CI: 0.73–0.99, p= 0.034). No genetic association was also detected between IFN-γR1 and SLE (OR= 0.97, 95% CI: 0.79–1.19, p= 0.768). Evidence from bidirectional MR did not support reverse causality. The weighted median regression also showed directionally similar estimates. Conclusion::Higher levels of IFN-γ are significantly associated with an increased risk of SLE, providing insights into the pathogenesis of SLE.

Combined detections of interleukin 27, interferon-γ, and adenosine deaminase in pleural effusion for diagnosis of tuberculous pleurisy

Background Previous studies reported interleukin-27 （IL-27）, interferon-y （IFN-γ）, or adenosine deaminase （ADA） alone plays a helpful role in diagnosing tuberculous pleural effusion （TPE）. The present study aims at comparing the diagnostic accuracy of pleural IL-27, IFN-γ, and ADA, and investigate the diagnostic accuracy of the combination of IL-27, IFN-γ, or/ and ADA for differentiating TPE from pleural effusions with the other etiologies. Methods The concentrations of IL-27, IFN-γ and ADA were simultaneously determined in pleural fluids and sera from 40 patients with TPE; 26 with malignant pleural effusion, seven with infectious pleural effusion, and eight with transudative pleural effusion by enzyme linked immunosorbent assay and colorimetric method. The corresponding biochemical indexs were also simultaneously determined. Results The concentrations of pleural IL-27 and IFN-γ in the tuberculous group were significantly higher than those in the malignant, infectious, and transudative groups. The concentrations of ADA in TPE were significantly higher than those in MPE or transudative effusions, while much lower than those in infectious effusions. Among these three biomarkers, IL- 27 was the most effective for TPE diagnosis, with the cut off value of 900.8 ng/L. IL-27 had a high sensitivity of 95% and specificity of 97.6% for differential diagnosis of TPE from non-TPEs. Combinations of IL-27, IFN-γ and ADA measurements further increased the sensitivity or specificity up to 100%. Conclusions Compared to non-TPEs, IL-27, IFN-γ and ADA all simultaneously increased in TPE; and among these three rapid detection methods, IL-27 appeared to be the best for distinguishing tuberculous from non-TPEs, especially from MPE. Combinations of the three markers （IL-27, IFN-γ and ADA） yielded the highest sensitivity and specificity. These findings suggest that the applications of a new biomarker, IL-27, alone or with IFN-γ and ADA, may contribute to more efficient diagnosis strategies in the management of tuberculous pleurisy.

Effects of Astragalus membranaceus in Promoting T-helper Cell Type 1 Polarization and Interferon-γ Production by Up-regulating T-bet Expression in Patients with Asthma

Objective： To explore the effect of Astragalus membranaceus （AM） on T-helper cell type 1 （Th1） specific transcription factor T-box expressed in T cells （T-bet） expression and Th1/Th2 equilibrium. Methods, The levels of T-bet mRNA in peripheral blood mononuclear cells （PBMCs） from 15 patients with asthma and 15 healthy subjects were determined by reverse transcription-polymerase chain reaction （RT- PCR）. PBMCs in asthma patients were incubated with AM and then the concentration of interferon γ （IFN-γ） and interleukin-4 （IL-4） in the supernate before and after AM intervention were determined by ELISA. The numbers of CD4 ＋ CCR3＋ and CD4＋CCR5＋ cells were counted by flow cytometry. Results： The expression of T-bet mRNA and the level of IFN-γ were lower, but level of serum IL-4 was higher in asthma patients when compared with those in healthy subjects respectively. After AM （60 μg/ml） intervention, the former two parameters raised and showed a positive correlation between them, while the level of IL-4 was decreased. The mean percentage of CD4＋CCR3＋ cells in asthma patients was significantly higher but that of CD4＋CCR5＋ cells was lower when compared with those in healthy subjects respectively. After AM intervention, the abnormal change in the two indexes was improved to certain extent, showing a reversing status of Th2 polarization. Conclusion： AM could increase the expression of T-bet mRNA and Thl cytokines such as IFN-γ, and might reverse the Th2 predominant status in asthma patients.

Adeno-associated virus mediated interferon-gamma inhibits the progression of hepatic fibrosis in vitro and in vivo

AIM:To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosis in vitro and in vivo. METHODS: We constructed the recombinant AAV encoding human INF-γ (rAAV- INF-γ) and took the primary rat hepatic stellate cells and carbon tetrachloride induced rats as the experimental hepatic fibrosis model in vitro and in vivo. Immunocytochemistry analysis was used to reveal the expression of α-SMA, the marker protein expressed in hepatic stellate cells. The mRNA expression of TGF-β, TIMP-1, and MMP-13 were analyzed by RT-PCR method. In vivo study, the hydroxyproline content in liver and serum AST, ALT were also detected. RESULTS: In vitro study, AAV vector could mediated efficient expression of human INF-γ, which inhibit the activation of hepatic stellate cells, decrease the expression of α-SMA and mRNA of TIMP-1, TGF-β, with the MMP-13 unchanged. In vivo study, the histological examination revealed that rAAV- INF-γ could inhibit the progression of the hepatic fibrosis. In the rAAV-INF-γ induced group, the hydroxyproline content and serum AST, ALT level were decreased to 177±28 μg/g wet liver, 668.5±140.0, 458.4±123.5 U/L, compare with the fibrosis control group 236±31 μg/g wet liver, 1 019.1±276.3, 770.5±154.3 U/L, respectively (P<0.01). mRNA expression of TIMP-1 in the rAAV-INF-γ induced rat liver was decreased while no significant change was observed in TGF-β and MMP-13. CONCLUSION: All these results indicated that rAAV-INF-γ has potential effects for gene therapy of hepatic fibrosis, which could inhibit the progression of hepatic fibrosis.

Interferon-γ Expression in Natural Killer Cells and Natural Killer T Cells Is Suppressed in Early Pregnancy

Recent study has suggested that innate immune system might play an important role in pregnancy progression. In this study, to investigate whether NK cells and NKT cells, instead of T cells, are the dominant populations of peripheral blood in early pregnancy, flow cytometry was used to detect the percentage and intracellular cytokine expressions of T cells, NK cells, NKT cdls in peripheral blood of non-pregnant women and early pregnant women. In our result, the percentages of NK calls and NKT calls were significantly increased in pregnancy compared to non-pregnancy. However, the percentage of T cells was not changed. We did not detect the Th2-dominance of total lymphocytes or T cells in peripheral blood of early pregnant women and there were also no significant changes of type 1 and type 2 cytokines in T cells, but IFN-γ production in both NK and NKT cells was decreased in early pregnancy. These results suggest that the innate immune system including NK cells and NKT cells should play a pivotal role in pregnancy progression. Type 1/type 2 shift mechanisms in innate immune system during the human early pregnancy should be paid more attention.

Effect of oxymatrine on interferon-gamma and tumor necrosis factor-alpha serum levels in an experimental rat model of autoimmune encephalomyelitis

BACKGROUND： Studies have demonstrated that experimental autoimmune encephalomyelitis （EAE） onset correlates with increased interferon-v （IFN-γ） and tumor necrosis factor-α （TNF-α） expression. Oxymatrine （OM） has been shown to inhibit autoimmune responses, but there are no reports showing that it could prevent the development of EAE. OBJECTIVE： To observe the effect of OM on serum levels of IFN-γ and TNF-α in a rat model of EAE.DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Experimental Animal Center of Henan Academy of Chinese Medicine and at the Key Disciplines Laboratory Clinical Medicine of Henan Province between July and December 2008. MATERIALS： OM was purchased from Chia-tai Tianqing Pharmaceutical, China; complete Freund＇s adjuvant was purchased from Sigma, USA. METHODS： Forty female Wistar rats were randomly assigned to four groups： EAE model （M）, low-dose OM treatment （OM-L）, high-dose OM treatment （OM-H）, and normal control （N, no immunization）, with 10 rats in each group. EAE was established in the M, OM-L, and OM-H groups following immunization with Guinea pig spinal cord homogenate and complete Freund＇s adjuvant. The M and N groups were intraperitoneally injected with normal saline （6.7 mL/kg per day）, the OM-L group received an intraperitoneal injection of OM （100 mg/kg per day）, and the OM-H group received OM （150 mg/kg per day）. MAIN OUTCOME MEASURES： At 16 days after immunization, the degree of histopathological changes in the spinal cord was assessed by hematoxylin-eosin stanining. Enzyme-linked immunosorbent assay was used to detect serum levels of IFN-γ, and radioimmunoassay was utilized to determine serum TNF-α level. Neurological scores were measured on a daily basis according to a 0-5 scale. RESULTS： Daily injections of OM, both high and low doses, resulted in decreased neurological scores in EAE rats （P〈0.01）, as well as reduced cellular infiltration in the spinal cord and decreased levels of serum IFN-γ and TNF-α （P〈 0.01）. CONCLUSION： OM reduced the onset and severity of EAE, which correlated with decreased IFN-γ and TNF-α expression.