Lack of interleukin-1α in Kupffer cells attenuates liver inflammation and expression of inflammatory cytokines in hypercholesterolaemic mice

BackgroundThe role of Kupffer cell interleukin (IL)-1 in non-alcoholic steatohepatitis development remains unclear.AimsTo evaluate the role of Kupffer cell IL-1α, IL-1β or IL-1 receptor type-1 (IL-1R1) in steatohepatitis.MethodsC57BL/6 mice were irradiated and transplanted with bone marrow-derived cells from WT, IL-1α−/−, IL-1β−/− or IL-1R1−/− mice combined with Kupffer cell ablation with Gadolinium Chloride, and fed atherogenic diet. Plasma and liver triglycerides and cholesterol, serum alanine aminotransferase (ALT), liver histology and expression levels of inflammatory genes were assessed.ResultsThe ablation and replacement of Kupffer cells with bone marrow-derived cells was confirmed. The atherogenic diet elevated plasma and liver cholesterol, reduced plasma and liver triglycerides and increased serum ALT levels in all groups. Steatosis and steatohepatitis were induced, but without liver fibrosis. A reduction in the severity of portal inflammation was observed only in mice with Kupffer cell deficiency of IL-1α. Accordingly, liver mRNA levels of inflammatory genes encoding for IL-1α, IL-1β, TNFα, SAA1 and IL-6 were significantly lower in mice with Kupffer cell deficiency of IL-1α compared to WT mice.ConclusionSelective deficiency of IL-1α in Kupffer cells reduces liver inflammation and expression of inflammatory cytokines, which may implicate Kupffer cell-derived IL-1α in steatohepatitis development.     

Effect of 17β-oestradiol on expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar cells

Oestradiol (E(2)) alters lymphocyte functionin vitro including T cell DNA synthesis and B cell immunoglobulin production in human tonsillar, splenic and peripheral blood cells. We have investigated whether one mechanism for this effect is that E(2) modifies the expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar mononuclear cells. Without E(2), addition of PHA (1 μg ml(-1)) for 10 h increased the expression of IL-2 and IL-6 mRNA but had no significant effect on IFN-γ mRNA. In separated T cells after 24 h incubation, E(2) (7×10(-8) M: ) increased only the IFN-γ mRNA levels. However, when E(2) was present in PHA-stimulated T cell cultures, mRNA levels from all cytokines were suppressed. E(2) decreased IL-2 mRNA levels in the T cell preparation after 24 h culture. For IL-6, E(2) decreased mRNA both in mononuclear cells and T cells after 10 h incubation. For IFN-γ, E(2) decreased mRNA levels in the mononuclear cell preparation after 24 h culture. Stimulation of the T cell preparation with PHA after 24 h incubation with E(2) decreased the IFN-γ mRNA levels compared to the cultures incubated with E(2) only. One part of the action of E(2) may be through a block in the up-regulation of the mRNA of IL-2, IL-6 and IFN-γ in activated cells.     

C/EBPβ and CHOP participate in Tanshinone IIA-induced differentiation and apoptosis of acute promyelocytic leukemia cells in vitro

Our studies indicated that Tanshinone IIA (TanIIA), which is widely applied in the treatment of cardiovascular diseases with a rare occurrence of side effects, could promote APL cell differentiation and apoptosis. We found TanIIA induced the differentiation of NB4 and MR2 cells with elevated C/EBPβ and CHOP. When C/EBPβ was overexpressed in NB4 cells, the level of CD11b in the transfected cells was significantly elevated. When we used CHOP siRNA to suppress CHOP expression in NB4 cells and then treated these cells with a high concentration of TanIIA, the differentiation and apoptosis of these cells were both significantly increased. These data demonstrate that C/EBPβ is critical for APL cell differentiation and apoptosis induced by TanIIA, and that CHOP acts as a negative regulator of C/EBPβ activity. Our study suggested that TanIIA is a promising drug for treating newly diagnosed and ATRA-resistant APL, and a high concentration of TanIIA associated with inhibition of CHOP, maybe a potentially promising therapy strategy.     

Down-regulating the expression of IL-3Rβ interfered with the proliferation,not differentiation in NB4 cells

The human IL-3 receptor is composed of both α and β subunits. In early studies, we showed that the level of IL-3Rβ expression was lower in patients with acute promyelocytic leukemia (APL) than healthy donors and patients in complete remission by real-time quantitative polymerase chain reaction (RT-qPCR). With the differentiation of cells, enhanced expression of IL-3Rβ was also observed in all-trans-retinoic acid (ATRA)-induced NB4 cells. To unravel the role of IL-3Rβ upregulation in NB4 cells induced with ATRA, we knocked down IL-3Rβ expression by RNA interference (RNAi). Knockdown of IL-3Rβ resulted in decreased proliferation in NB4 cells induced with or without ATRA, observed by cell growth curves, colony formation assays and cell cycle analysis. Surface expression of CD11b antigen and nitroblue tetrazolium (NBT) reduction assays were also carried out at different time points. However, no significant difference was observed between the experimental and control groups treated with ATRA. Other findings suggested that IL-3Rα was decreased in NB4-IL-3Rβ shRNA cells by western blot. Down-regulation of IL-3Rβ also caused a decrease in PML/RARα expression detected with RT-qPCR. Together, these results suggest that abnormalities of IL-3Rβ expression were observed in APL; knockdown of IL-3Rβ inhibited the proliferation of NB4 cells with or without ATRA, but no effect was detected in the cellular differentiation. When NB4 cells exposed to ATAR, the up-regulation of IL-3Rβ expression may contribute to the maintenance of proliferation rather than cell differentiation.     

Low expression of costimulatory molecules and mRNA for cytokines are important mechanisms of immunosuppression in acute lymphoblastic leukemia in children?

Mechanisms leading blasts of acute lymphoblastic leukemia to escape from immune surveillance are still unknown. Only few reports showed that ALL cells are inefficient antigen presenting cells. The aim of the study was to assess expression of critical costimulatory/adhesion molecules and mRNA for main pro- and anti-inflammatory cytokines in ALL cells. Children with B-cell precursor ALL (n=20) were prospectively enrolled into the study. Expression of costimulatory/adhesion molecules (CD1a, CD11c, CD40, CD54, CD80, CD83, CD86, CD123, HLA class I and II) was assessed by flow cytometry and mRNA for cytokines (IFN-gamma, IL-10, IL-4, TGF-beta) - with real-time PCR. Results: 1) high expression was observed for HLA I and II class, moderate for CD40, CD83, CD86 and low or no expression for CD80, CD54, CD1a, CD11c and CD123; 2) we found expression of mRNA for IFN-gamma, IL-10, IL-4 and TGF-beta in blasts cells (but not in all specimens). We noted relatively lower expression of all assessed cytokines comparing to T-cells obtained from healthy donors but interestingly expression for IL-10 was higher in normal B-cells than in blast cells, and IFN-gamma and IL-4 were not found in normal B-cells. In summary we suggest that ALL-blasts present low expression of costimulatory/adhesion molecules and mRNA for cytokines and this probably contribute to the absence of host T- cells stimulation to immune response.     

Construction of the pIRES2-ZsGreen1 eukaryotic expression vector of factor Ⅸ gene and expression in HEK-293 cells

正Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using the     

Effect of vitamin E succinate on expression of TGF-β_1, c-Jun and JNK1 in human gastric cancer SGC-7901 cells

Vitamin E succinate(RRR-α-Tocopheryl Succinate,VES), a derivative of natural vitamin E, is acompound esterified by succinic acid and 6-hydroxyl-α-tocopheryl. VES can increase thestability of α-tocopheryl and protect 6-hydroxylfrom oxidation. Nowadays, this kind of vitamin Ehas been on sale on the markets. Previous researchesindicate that VES has an evident inhibitory effecton the growth of various tumor cells, but has noharm to the growth of normal cells. Theexperiment in vivo has demonstrated that VES can     

Influence of IFNα-2b and BCG on the release of TNF and IL-1 by Kupffer cells in rats with hepatoma

!NTRODUCTIONKuPffer eells are residential macroPhages in the1 iver,which Play a eritieal role in the maintenaneeof normal liver function and in immunal surveilaneeof hePatoeellular earcinoma(HCC)and othereaneers〔l].The bio一ogieal immune modulants havebeen used for treating Patients with HCC and othereaneers[2〕.In our previous studies,the eombineduse of biological immune modulants showed bettereffeets.The normal rats and hePatoma rats indueedby DEN(Diethylnitrosamine)were treated …     

Expansion of CD4+CD25+ regulatory T cells from cord blood CD4+ cells using the common ��-chain cytokines (IL-2 and IL-15) and rapamycin

Rapamycin has important roles in the modulation of regulatory T cells. We tried to expand CD4(+)CD25(+) regulatory T cells (Treg cells) from umbilical cord blood (CB) CD4-positive cells using interleukin (IL)-15 or IL-2 with transforming growth factor (TGF)-β and rapamycin. We were able to obtain more than 500-fold expansion of CD4(+)CD25(+) cells from CB CD4(+) cells using IL-15 and TGF-β with rapamycin. These expanded CD4(+)CD25(+) cells expressed forkhead box P3 (FoxP3) mRNA at a level about 100-fold higher and could suppress allogeneic mixed lymphocyte culture (MLC) by more than 50%. Early after rapamycin stimulation, CB CD4(+) cells showed increased expression of FoxP3 and a serine/threonine kinase Pim2 and sustained expression of negative phosphoinositide 3-kinase regulator phosphatase and tensin homolog deleted on chromosome 10 (PTEN). On the other hand, CD4(+)CD25(+) cells expanded with rapamycin for 8 days showed much higher levels of FoxP3 mRNA expression and decreased expression of PTEN. A comparison of IL-15 stimulation and IL-2 stimulation showed slightly higher efficiency of IL-15 for expansion of CD4(+)CD25(+) cells, and for FoxP3 expression, IL-15 also showed significantly higher efficacy for inhibition of MLC. The combination of the common γ-chain cytokine IL-15, TGF-β, and rapamycin may be a useful means for expanding Treg cells. Pim2 expression early after stimulation with rapamycin may be important for conferring rapamycin resistance for growth of Treg cells. IL-15 is not less useful than IL-2 for expansion of Treg cells.     

Effect of hypoxia on expression of placental trophoblast cells SATB1 and β-catenin and its correlation with the pathogenesis of preeclampsia

Objective: To study the effect of hypoxia on the expression of placental trophoblast cells SATB1 and β-catenin and its correlation with the pathogenesis of preeclampsia. Methods: Trophoblastic cell lines HRT8/SVneo were cultured, SATB1 and β-catenin expression and cell biological behavior were determined after hypoxia reoxygenation treatment; cell biological behavior and the expression of related genes were determined after the transfection of SATB1 and β-catenin siR NA; preeclampsia placenta and normal placenta tissues were collected and the expression of SATB1 and β-catenin were determined. Results: OD value, cell migration rate, m RNA contents of SATB1 and β-catenin of H/R group were significantly lower than those of Nor group, cell apoptosis rate was higher than that of Nor group and the number of invasive cells was less than that of Nor group; OD value and bcl-2 mRNA content of SATB1-siRNA group were lower than those of NC group; cell apoptosis rate as well as Bax, Caspase-3, caspase-6 and caspase-9 mRNA contents were higher than those of NC group; cell migration rate as well as CTSB, CTSD, MMP2 and MMP9 mRNA contents of β-catenin-siRNA group were lower than those of NC group; the number of invasive cells was less than that of NC group; the expression levels of SATB1 and β-catenin in preeclampsia placenta tissue were significantly lower than those in normal placenta tissue. Conclusions: Hypoxia can inhibit the expression of SATB1 and β-catenin in the pathogenesis of preeclampsia, which can affect the proliferation, apoptosis, migration and invasion of cells.     

Establishment and expression of recombinant human glial cell line-derived neurotrophic factor and TNF α receptor in human neural stem cells

ObjectiveTo investigate the interference and expression of human glial cell line-derived neurotrophic factor (hGDNF) and soluble TNF alpha (sTNFR |) receptor genes in neural stem cells and to evaluate the roles of these proteins in the genetic treatment of spinal cord injury.MethodsFull-length of GDNF cDNA (558 bp) and sTNFR|cDNA (504 bp) were inserted into the early 1 region of adenovirus genomic DNA respectively and were immediated by the human cytomegalovirus (gene promoter/enhancer). These adenoviruses were propagated in HEK293 cells via homologous recombination for 7-10 days in vivo, then they were used to infect human neural stem cells. The infection and expression of gene were tested under immunofluorescence, ELISA and Western-blot after 48 hours.ResultsAlmost all the cultured cells showed the nestin immunofluorescence positive staining, which was the characteristics of neural stem cell. A great quantity of EGFP and RFP were observed in neural stem cells, which indicated the expression of GDNF and sTNFR |. After transfection of GDNF and sTNFR | genes, many neural stem cells show GFAP and tubulin immunofluorescence positive staining, which meant that most neural stem cells differentiated into neuron at that condition.ConclusionsThe infective efficiency of adenovirus is greatly acceptable to neural stem cell, thus adenovirus provide a useful vector for exogenous GDNF and sTNFR | genes expressing in neural stem cells, which is useful for differentiation of neural stem cell.     

Effects of 17β-estradiol on the expression of matrix metalloproteinase-1, -2 and tissue inhibitor of metalloproteinase-1 in human osteoblast-like cell cultures

Estrogen can effectively prevent estrogen deficiency-induced bone loss in animals and humans. However, its mechanism remains unknown. Osteoblast-derived Matrix metalloproteinse-1 (MMP-1), MMP-2, and tissue inhibitor of metalloproteinase-1 (TIMP-1) recently were implicated as playing important roles in initiating bone resorption. Therefore, we tested the effects of 17β-estradiol (E₂) on MMP-1, MMP-2, and TIMP-1 production in cultures of human osteoblastic MG-63 cells and normal human osteoblasts (hOB). MMP-1, MMP-2 and TIMP-1 concentrations in the culture medium were determined by ELISA, and activity of MMP-2 was assessed by ELISA. After 12–48 h of treatment, E₂ at 10⁻⁸M decreased MMP-1 level in cultures of MG-63 cells or hOB. Treatment with increasing dose of E₂ in MG-63 cells or hOB caused a dose-dependent decrease in MMP-1 synthesis. E₂ had no influence on MMP-2 and TIMP-1 production in MG-63 cells or hOB cultures, as well as activation of latent MMP-2. In conclusion, E₂ represses MMP-1 synthesis, and this effect may contribute to its action on the inhibition of bone resorption, followed by prevention of bone loss. Increasing MMP-1 production followed by estrogen deficiency may contribute to the mechanisms involved in postmenopausal osteoporosis.