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Hepatic ischemia-reperfusion (I/R) injury, a common clinical complication of liver transplantation, gravely affects patient prognosis. Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins. KLF6, a member of the KLF family proteins, plays crucial roles in proliferation, metabolism, inflammation and injury responses; however, its role in HIR largely remains unclear. Herein, we found that KLF6 expression was significantly up-regulated in mice and hepatocytes after I/R injury. Subsequently, mice were subjected to I/R after tail vein injection of shKLF6- and KLF6-overexpressing adenovirus. KLF6 deficiency markedly aggravated liver damage and cell apoptosis along with the activation of hepatic inflammatory responses, whereas hepatic overexpression of KLF6 in mice showed opposite effects. Furthermore, we knocked out or overexpressed KLF6 in AML12 cells, and then exposed to hypoxia-reoxygenation challenge. KLF6 knockout significantly reduced cell viability, and increased hepatocyte inflammation, apoptosis, and ROS, whereas overexpression of KLF6 showed the opposite effects. Mechanistically, KLF6 inhibited the overactivation of autophagy at the initial stage, and the regulatory effect of KLF6 on I/R injury was autophagy-dependent. CHIP-qPCR and luciferase reporter gene assays confirmed that KLF6 was bound to the promoter region of Beclin1 and inhibited its transcription. Moreover, KLF6 activated the mTOR/ULK1 pathway. Finally, we retrospectively analyzed the clinical data of liver transplantation patients and observed significant associations between KLF6 expression and liver function after liver transplantation. In summary, KLF6 inhibited the overactivation of autophagy by transcriptional regulation of Beclin1 and activation of the mTOR/ULK1 pathway, thereby playing a protective role against hepatic I/R injury. KLF6 is expected to serve as a biomarker to predict the severity of I/R injury after liver transplantation.

We investigated the effect on rheumatoid arthritis (RA) of an anti-gp130 monoclonal antibody (mAb) and its mechanism using RA fibroblast-like synoviocytes (FLS) and a collagen antibody-induced arthritis (CAIA) mouse model. We determined the interleukin 6 (IL-6), IL-6 receptor α (IL-6Rα), gp130, receptor activator of nuclear factor κB ligand (RANKL), matrix metalloproteinase 3 (MMP3), TIMP metallopeptidase inhibitor 1 (TIMP1), and Bcl-2 levels in RA and osteoarthritis (OA) serum and synovial fluid. RA FLS were cultured with or without IL-6/IL-6Rα; WNT5A and RANKL levels were detected. We generated an anti-gp130 mAb (M10) with higher affinity and specificity, blocked IL-6 signaling with it, and assessed its effects on the CAIA model, WNT5A and RANKL expression, and signal transducer and activator of transcription 3 (STAT3) phosphorylation. The IL-6 signaling system in patients with RA was increased; RANKL, MMP3, TIMP1, and Bcl-2 in RA bone were elevated. IL-6/IL-6Rα increased RA FLS WNT5A and RANKL expression. M10 ameliorated arthritis in the CAIA model, and inhibited RANKL, WNT5A, and Bcl-2 expression in RA FLS by blocking IL-6 signaling, likely via Janus kinase-STAT3 pathway downregulation. The IL-6-soluble IL-6Rα-gp130 complex is hyperactive in RA and OA. M10 may be the basis for a novel RA treatment drug.

Metastases are the greatest contributors to death from breast cancer. Here, we identified a distinct subpopulation of luminal breast cancer characterized by cytokeratin 14 (CK14) expression in secondary colonies rather than primary tumors. This entity possessed a poorer prognosis compared to their CK14(-) counterparts. Immunohistochemical analysis showed that myeloid-derived suppressor cells (MDSCs) were recruited into the tumor microenvironment and exhibited a close spatial relationship with CK14(+) cancer cells. We demonstrated that histidine decarboxylase (Hdc) is capable of labeling myeloid-biased hematopoietic stem cell/progenitor cell (HSC/HSPC) and immature myeloid cells infiltrating in tumor tissues. FACS data obtained from Hdc-CreER(T2); eGFP; MMTV-PyVT female mice revealed an increased percentage of Hdc(+) PMN-MDSCs in metastatic masses. Hdc(+) PMN-MDSCs expressed high levels of canonical Wnts, including Wnt2, Wnt4, Wnt5a, and Wnt7b, to aberrantly activate Wnt/β-catenin signaling in CK14(+) malignant cells. β-catenin translocated from the membrane into the cytoplasm and nucleus. Targeted ablation of Hdc(+) PMN-MDSCs-derived Wnts through porcupine(flox/flox) and iDTR transgenic models hampered the metastatic cascade, making Hdc(+) immature myeloid cells an attractive candidate for tailed immunotherapies.

An unprecedented Rauhut-Currier-type 1,6-conjugate addition has been developed. With chiral cyclohexane-based phosphine-amide catalyst 3 h, the 1,6-conjugate reaction has been achieved to produce chiral diarylmethine compounds in excellent yields (91-99 %) and enantioselectivities (92-98 % ee).

To study the interaction between sleep deprivation and immune functions in mice.To set up the uncompleted sleep deprivation of mice by means of lighting mimicked with whole day and dark box done with whole night for 2 weeks and to deprive the mice of sleep in the rotating cage for 24 h and 72 h. All mice are challenge with BSA before sleep deprivation. The spleen weight of mice was measured by analytic balance. The count of T subpopulation was detected with FACS. The levels of cytokines IL-2, IL-10 and the concentration of the specific antibody to BSA was detected by ELISA method.Compared with the normal control, the spleen weight of mice in all the other experimental groups was decreased (P0.05) except those of lighting group. Decrease of CD8+ T lymphocyte was observed (P0.01) while the ratio CD4/CD8 increased greatly. Augmentation of IL-10 was demonstrated only in the all-light group, both IL-2 and IL-10 decreased (P0.01) in the other groups. Significant decrease of specific antibody for BSA was found in the mice of sleep deprived group (P0.01).Complete and incomplete sleep deprivation inhibit differently the immune function of mice by triggering decrease of CD8+ T cells, inhibiting secretion of specific antibody and causing disturbance on the pattern of cytokines.

To observe the effect of electroacupuncture (EA) and moxibustion of "Dazhui" (GV 14) on the proliferation levels of the splenetic CD4+ CD25+ regulatory T cells (Tregs) of H22 tumor-bearing mice in vitro.Forty eight Balb/c mice were randomized into control, model, moxibustion and EA groups, with 12 cases in each. H22 tumor-bearing model was set up by hypodermic injection of H22 tumor cells (0.2 ml, 1 x 10(7) cells/ml). EA (2 Hz, 2 mA) was applied to "Dazhui" (GV 14) and left "Huantiao" (GB 30) for 20 min, and moxibustion was applied to "Dazhui" (GV 14) 2 moxa-cones every time. The treatment was given from the 2nd day on after innoculation of tumor cells, once every other day, 6 times altogether. After the treatment, the mice were killed by peeling off the eyeball and blood samples were collected to be separated into serum. Then, Tregs of the spleen tissues of Balb/c mice in different groups were isolated by using megnetic activated cell sorting (MACS) system to be cultured independently, and co-cultured with EA-treated serum and moxibustion-treated serum separately in culture fluid for 96 h, added with 3H-tritiate thymidine (TdR) in the culture-fluid 12 h before the end of culture, followed by collecting the cells and detecting their proliferation levels (count per minute, cpm) by using a lipid scintillation device.The proliferation level of Tregs in model group was elevated significantly compared to normal control group (P0.05), while in comparison with model group, those of Tregs of EA and moxibustion groups decreased considerably (P0.01). After separate application of the diluted acupuncture-treated serum and moxibustion-treated serum at 1 : 1 and 1 : 8 (not 1 : 16 and 1 : 32) to the cultured Tregs, their proliferation levels (cpm) in EA and moxibustion groups were obviously upregulated in comparison to those of normal control group (P0.05), and the cpm in EA group was significantly higher than that in model group (P0.05), suggesting a different action mechanism between acupuncture-moxibustion treatment and serum stimulation.EA of "Dazhui" (GV 14) and "Huantiao" (GB 30) and moxibustion of "Dazhui" (GV 14) can effectively downregulate the proliferation level of the cultured splenetic Tregs of the tumor bearing mice. EA-treated serum and moxibustion-treated serum diluted at 1 : 1 and 1 : 8 can evidently upregulate the proliferation level of Tregs in vitro.

Metabolism is the set of chemical reactions that occur in living organisms in order to maintain life, and a genome-scale metabolic network can be reconstructed by identifying, categorizing and interconnecting all the genes, proteins, reactions and metabolites that participate in the metabolic activity of a biological system to form a metabolic network. Enzymes play a very important part in metabolism, and the identification of all genes encoding metabolic enzymes and to assign correct Enzyme Commission classification (EC) numbers to them is pivotal in the reconstruction of metabolic networks. In this paper, we represent an automated and efficient gene-enzyme identification method in the reconstruction of metabolic networks: Hybrid Participle Algorithm (HPA). In order to prove the usefulness of HPA, we reconstructed the metabolic networks of Escherichia_coli_K12 using both PathoLogic and HPA. The results indicate that by using HPA, we can identify more metabolic genes and their corresponding enzymes than by using other methods based on the whole name match method, such as PathoLogic. And the F-measures of our results are higher than the F-measures of the results obtained by using PathoLogic. HPA provides an automated and efficient way to identify the metabolic genes and their corresponding enzymes.

To study on the role of IFN-gamma, IL-10, IL-12 and TRAIL in the pathogenesis of RA.The concentrations of IFN-gamma, IL-12, IL-10 and TRAIL in synovium fluid and sera isolated from 46 patients with RA were determined by Sandwich ELISA.The concentrations (ng/L) of IFN-gamma, IL-12 and TRAIL in synovium fluids from the patients were (106.2+/-7.8), (57.7+/-3.5) and (166.5+/-12.3), respectively. The concentrations (ng/L) of these cytokines in sera from the patients were (56.3+/-7.4), (35.1+/-12.7), (69.5+/-8.3) and in sera from the healthy donors (31.1+/-4.4), (25.2+/-2.6) and (41.2+/-4.8). The levels of cytokines mentioned above in synovium fluid from the patients were higher than those in sera from both the patients and the healthy individuals (P0.05, P0.01). There was no significant difference of concentration of IL-10 between the patients and the healthy donors.The levels of IFN-gamma, IL-12 and TRAIL in synovium fluid from the patients with RA are higher than those in healthy donors. This result indicates that the pattern of cytokine on course of RA is main of Th1, more typical in synovium fluid than in serum. Analysis of the concentration of IFN-gamma, IL-12, IL-10 and TRAIL in the serum may be useful to diagnosis and bio-immunological treatment of the RA patients correctly.

To express recombinant human FasL molecule in E.coli.RT-PCR was applied to amplify FasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. The DNA fragment was cloned into PCR2.1 vector. After sequencing, the FasL gene was inserted into pQE-31 vector and expressed in E.coli M15. The FasL protein was purified through Ni-ATA affinity chromatography column and identified by SDS-PAGE and Western blot. The mice were immunized with the FasL protein and the specific anti-serum was harvested 6 weeks after immunization. The serum level of FasL from with different kinds of diseases patients were detected using the anti-FasL antibodies from the immunized mice.The expressed protein could be recognized by anti-human FasL antibody in Western-blot analysis with M(r)40 000. This protein could induce Jurket cells apoptosis. anti-FasL serum prepared from mouse could detect the serum FasL as sensitive as commercial ELISA kits.The human FasL protein is obtained. It lays the foundation for the further detecting the concentration of FasL and sFasL of patients.

To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report. rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above. The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA. The molecular weight and specificity were demonstrated by PAGE and Western blot. The density of the specific precipitation bands was determined by gel scanning. The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI). The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant. The molecular weight was 22 kD. The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively. The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively. The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L. The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC. The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity. The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale.

To prepare monoclonal antibody specific to P-selectin lectin-EGF domain, the gene for lectin-EGF domain of P-selectin L-EGF was amplified from normal human platelets by RT-PCR, then was cloned into prokaryotic vector pET42b(+). The recombinant plasmid was transformed into E. coli DH5 alpha strain for further screening and characterization, and was expressed in E. coli BL21 strain. Expressed protein was purified by chromatography on a Ni(2+)-NTA superflow agarose column and eluted with pH 8.0-4.5 urea gradient. Then the mAb anti-lectin-EGF was prepared with classical hybridoma technique, and 3 hybridoma cell lines (B10, F3 and H5) were obtained with Ig subclasses of these mAbs were IgG(2), IgG(1), and IgG(3) respectively, and their light chains were all kappa chain. Immuofluorescence and FACS assays demonstrated that mAbs could specifically recognize P-selectin expressed on ECV (endothelial cell line) stimulated by LPS. Meanwhile, the role of mAbs to P-selectin lectin-EGF domain was studied, and it was proved that the mAbs markedly inhibited adhesion between platelets and neutrophils in vitro. These monoclonal antibodies can specifically recognize the natural P-selectin and markedly inhibit adhesion between platelets and neutrophils in vitro.

AIM: To investigate the potential role of Active Chinese mistletoe lectin-55 (ACML-55) in tumor immune surveillance. METHODS: In this study, an experimental model was established by hypodermic inoculating the colon cancer cell line CT26 (5 × 105 cells) into BALB/c mice. The experimental treatment was orally administered with ACML-55 or PBS, followed by the inoculation of colon cancer cell line CT26. Intracellular cytokine staining was used to detect IFN-γ production by tumor antigen specific CD8+ T cells. FACS analysis was employed to profile composition and activation of CD4+, CD8+, γδ T and NK cells. RESULTS: Our results showed, compared to PBS treated mice, ACML-55 treatment significantly delayed colon cancer development in colon cancer -bearing Balb/c mice in vivo. Treatment with ACML-55 enhanced both Ag specific activation and proliferation of CD4+ and CD8+ T cells, and increased the number of tumor Ag specific CD8+ T cells. It was more important to increase the frequency of tumor Ag specific IFN-γ producing-CD8+ T cells. Interestingly, ACML-55 treatment also showed increased cell number of NK, and γδT cells, indicating the role of ACML-55 in activation of innate lymphocytes. CONCLUSION: Our results demonstrate that ACML-55 therapy can enhance function in immune surveillance in colon cancer-bearing mice through regulating both innate and adaptive immune responses.