Identification and characterization of the CD226 gene promoter.

CD226 is one of the main activating receptors on natural killer cells, and it can induce cytotoxicity to target cells through interaction with its ligands CD155 or CD112. CD226 is also involved in T cell differentiation, activation, and cytotoxicity. The expression of CD226 on natural killer cells and T cells can be regulated by cytokines and chemical stimuli; however, the mechanism of the regulation of the CD226 gene is still unknown. In this study, we have identified two promoters in the human CD226 gene named P1 and P2, which are located at -810 to -287 bp and +33 to +213 bp, respectively, and a negative regulation element between P1 and P2. Both P1 and P2 can be regulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and calcium ionophore (A23187). Bioinformatics analysis shows that, within this CD226 gene region, there are putative binding sites for transcription factors AP-1, Sp1, PEA3, and Ets-1. We have found that transcription factor activating protein-1 (AP-1) can up-regulate CD226 promoters P1 and P2 in human hepatocarcinoma cells, a hepatocarcinoma cell line with low expression of endogenous AP-1 and Ets-1. Interestingly, the transcription factor Ets-1 promotes AP-1-induced P2 activity but inhibits AP-1-induced P1 activity for which a 10-bp AP-1/Ets-1 composite site (CCTTCCTTCC) in P1 may be responsible.

Dynamic changes of apoptosis-inducing ligands and Th1/Th2 like subpopulations in Hantaan virus-induced hemorrhagic fever with renal syndrome.

The expression of the apoptosis-inducing ligands, TNF-alpha, FasL and TRAIL on peripheral blood mononuclear cells (PBMC) and the levels of their soluble form (TNF-alpha, sFasL and sTRAIL) in plasma from 40 hemorrhagic fever with renal syndrome (HFRS) patients as well as 26 healthy blood donors were determined by flow cytometry (FCM) analysis and sandwich ELISA, respectively. The status of Th1, Th2, Tc1 and Tc2 subsets in PBMC was evaluated by intracellular cytokine staining and FCM. Compared to controls, the expression of membrane bound FasL and TRAIL was up-regulated on surface of PBMC isolated from the HFRS patients, particularly on CD8+ T lymphocytes. The levels of TNF-alpha, sFasL and sTRAIL in plasma from the HFRS patients in the acute phase increase 4.7-fold, 6.0-fold and 1.8-fold, respectively, over those from the healthy donors. The percentage of Th1, Tc1 and Tc2 subsets in PBMC from the patients also increased significantly compared with those from healthy donors. These results indicate that dynamic changes occurred in both the membrane bound and soluble forms of apoptosis-inducing ligands (FasL, TRAIL and TNF-alpha) and proportions of Th1 and CTL in HFRS patients increased. Both factors may play an important role in the etiology of Hantaan virus infection in humans.

Generation of monoclonal antibodies to cancer/testis (CT) antigen CT10/MAGE-C2.

CT10/MAGE-C2 is a recently identified antigen that, typically of cancer/testis (CT) antigens, can be found in various malignant tumors and in normal adult testis. As with many other CT antigens, our knowledge is based mainly on mRNA expression data. In the present study, we describe the generation of mAbs to CT10/MAGE-C2 for the analysis of its protein expression. Newly generated clones were chosen based on their reactivity in ELISA, immunoblotting, and immunohistochemistry (IHC). Emphasis was put on the reactivity of newly generated reagents on formalin-fixed, paraffin-embedded tissue to ensure their applicability to archival material. Eventually we selected two clones, LX-CT10.5 and LX-CT10.9, that showed intense reactivity to CT10/MAGE-C2 protein and CT10/MAGE-C2 mRNA-positive cell lines, but no cross-reactivity with other CT antigens. Both mAbs show superior staining characteristics in IHC and are applicable to frozen and paraffin sections. In testis, CT10/MAGE-C2 displays the typical CT pattern with regard to staining of germ cells, which is intense during the early maturation stages. In tumors, we analyzed a limited number of cases displaying the typical heterogeneous CT expression pattern. Interestingly, immunoreactivity was seen solely in the nucleus: No staining was seen in the cytoplasm of tumor cells.

[Identification of cell lines expressing the putative ligand(s) for LAIR-1(CD305) and LAIR-2(CD306)].

AIM: To identify cell lines expressing the putative ligand(s) for LAIR-1 and LAIR-2. METHODS: CHO cell lines secreting LAIR-1-Fc or LAIR-2-Fc fusion protein were prepared and the supernatants from the CHO cell lines were collected and purified by protein A affinity chromatography column. Several cell lines were stained with the purified LAIR-1-Fc and LAIR-2-Fc fusion proteins and then analyzed by flow cytometry for detecting the expression of their putative ligand(s). To exclude non-specific binding between the cells and the fusion proteins, the cell lines expressing their putative ligand(s) were pre-incubated with the mAbs against LAIR-1 or LAIR-2, stained with the fusion proteins, and analyzed by flow cytometry. RESULTS: Both LAIR-1 and LAIR-2 fusion proteins bound strongly with human amnion-derived epithelial cell line WISH, and moderately with human melanoma cell line C32, human embryo kidney epithelial cell line 293T, and human umbilical vein endothelial cell line ECV304. Furthermore, this binding could be blocked by the mAb FMU-LAIR-2.2 (recognizing both LAIR-1 and LAIR-2) or mAb FMU-LAIR-2.1(recognizing LAIR-2). CONCLUSION: The putative ligand(s) for LAIR-1 and LAIR-2 were expressed on WISH, C32, 293T and ECV304 cell lines. LAIR-1 and LAIR-2 may share the same ligand(s). These findings lay the foundation for the molecular cloning of the putative ligand(s) for LAIR-1 and LAIR-2.

[Changes and significance of TNF, sIL-2R, IL-6, IL-4 and IFN-gamma levels in plasma from the patients with hemorrhagic fever with renal syndrome].

AIM: To explore the changes of TNF, sIL-2R, IL-6, IL-4 and IFN-gamma levels in plasma from patients with hemorrhagic fever with renal syndrome(HFRS) and analyze the correlation between these cytokine levels and alanine aminotransferase(ALT) level in sera from the patients. METHODS: The cytokine levels were measured by double mAb sandwich ELISA. An automatic biochemical detector(RA-1000) was employed to determine the ALT level. RESULTS: Levels of TNF, IL-6, IL-4, IFN-gamma and sIL-2R in plasma from the patients were(95.82+/-12.04), (362.46+/-141.26), (17.76+/-3.52), (116.18+/-19.80) ng/L and (898 820+/-127 200) U/L, respectively. These cytokines in plasma from healthy donors were (17.89+/-1.68), (43.81+/-18.08), (4.86+/-1.14), (7.57+/-2.41) ng/L and (66 730+/-29 690) U/L, respectively. The cytokine levels of the patients were notably higher than those of the healthy donors.(P<0.01). At the same time, the average ALT level in sera from the patients was 4.4 fold of that from the healthy donors. Statistical analysis showed that levels of TNF, sIL-2R, IL-6 and IFN-gamma were positively correlated with ALT level, respectively. CONCLUSION: The HFRS patients have a significant increase in both cytokines (TNF, sIL-2R, IL-6 and IFN-gamma) and ALT levels. And there is a positive correlation between those cytokine and ALT levels, suggesting that the liver injury during HTNV infection may be related to the elevation of above cytokine levels.

[Construction and expression of human EpCAM eukaryotic expression vectors and identification of their products].

AIM: To construct EpCAM eukaryotic expression vectors pIg-EpCAM and pEGFP-EpCAM and to express them in COS7 cells. METHODS: EpCAM cDNA was amplified by PCR and then inserted into the pIg and pEGFP vectors respectively to construct recombinant vectors pIg-EpCAM and pEGFP-EpCAM. The two recombinant vectors were transfected into COS7 cells under the mediation of liposome. The expressed EpCAM-Ig fusion protein was detected by Western blot. The expression of EpCAM-GFP fusion protein was observed under fluorescence microscope. RESULTS: DNA sequencing demonstrated that EpCAM was correctly cloned into the two vectors. The culture supernatant of the pIg-EpCAM-transfected COS7 cells could bind effectively to mAb against human IgG Fc. Green fluorescence distributed evenly in the cytoplasm and nuclei of pEGFP-transfected COS7 cells, whereas in pEGFP-EpCAM transfected COS7 cells, green fluorescence distributed mainly on the surface of the cells. CONCLUSION: Two EpCAM eukaryotic expression vectors have been constructed and expressed successfully, which lays the foundation for functional research of EpCAM and for preparation of mAb to EpCAM.

[The inhibition of TNF-induced NF-kappaB nuclear translocation in ECV304 cells by mAbs against human TNF].

AIM: To identify the inhibition of TNF-induced NF-kappaB nuclear translocation by three anti-human TNF mAbs, D2, E6 and F6. METHODS: TNF solutions were pretreated with mAbs D2, E6 and F6 as well as control mAb at 37 degrees Celsius for 1 h, respectively, and then they were added to ECV304 cell cultures. After 1 hour, the cells were harvested and nuclear proteins were extracted. The NF-kappaB activity in nuclear extract was detected by electrophoretic mobility shift assay (EMSA). RESULTS: All of the three anti-TNF mAbs could inhibit TNF-induced NF-kappaB nuclear translocation in a dose-dependent manner. At the concentrations of 10 mg/L and 0.1 mg/L, the inhibition rates of mAb D2, E6 and F6 were 94.2% and 75.1%, 64.9% and 28.6%, 70.3% and 49.5% respectively, while the inhibition rate of control mAb was only 20.0% and 11.1%. CONCLUSION: mAbs D2, E6 and F6 can specifically inhibit TNF-induced NF-kappaB nuclear translocation, which lays the foundation for preparation of therapeutic chimeric anti-human TNF antibody for treatment of infectious and autoimmune diseases.

[Cloning, prokaryotic expression of LAIR and identification of their immunological reactivities].

AIM: To clone and express the leukocyte-associated immunoglobulin-like receptor (LAIR) and to identify the immune reactivity of LAIR-2 to anti-LAIR-1 specific monoclonal antibodies(mAb). METHODS: Genes encoding LAIR-1 and LAIR-2 were cloned by RT-PCR from human peripheral blood mononuclear cells(PBMC) and two leukemia cell lines Jurkat and HL-60. The extracellular region gene of LAIR1 and LAIR-2 were inserted into vector pGEX-4T-3 expressing GST fusion protein, expressed on IPTG induction and purified through glutathione-sepharose 4B column. The immunological reactivity of expressed LAIR-2 to anti-LAIR-1 mAb was identified by indirect ELISA. RESULTS: LAIR-1 and LAIR-2 cDNAs had been cloned and expressed. Five new LAIR-1 cDNA isoforms were cloned. Among them, two isoforms from HL-60 included LAIR-1 open reading frames (ORF) and three isoforms from Jurkat were LAIR-1 cDNA segments. The LAIR-1 and LAIR-2 showed different immunological reactivities. CONCLUSION: The transcription, processing after transcription and expression of LAIRs may be related to disparities in individuals and disease status. The difference in immunological reactivity may be involved in their structure.

[Effects of perindopril on plasma soluble TRAIL and receptor soluble DR5 in patients with congestive heart failure].

AIM: To explore the levels of soluble TRAIL and DR5 in plasma of patients with congestive heart failure (CHF) and the effects of perindopril. METHODS: 58 CHF patients were randomly divided into two groups, namely perindopril treatment group (30 cases) and routine treatment group (28 cases). The levels of sTRAIL and sDR5 in plasma of 30 CHF patients treated with perindopril, 28 CHF patients treated with routine method before and after treatment and 20 healthy persons were detected by ELISA. RESULTS: (1)The mean levels of sTRAIL in plasma of 58 CHF patients and 20 healthy persons were (1.43+/-0.47) microg/L and (0.93+/-0.12) microg/L, respectively, the comparison between the patients and healthy persons had no notable difference (P>0.05), suggesting that plasma sTRAIL level had no significant relation to injured level of cardiac function. As for sDR5 level, the mean level in plasma of 58 CHF patients was (39.67+/-6.78) ng/L, this value was significantly higher than that of healthy control group, and the level of sDR5 was increased with the injured level of cardic function.(2)The plasma levels of sTRAIL in both perindopril group and conventional treatment group decreased after treatment, but there was no significant difference. The mean levels of plasma sDR5 in perindopril group were (31.23+/-10.16 ) ng/L and (8.50+/-2.14) ng/L; the levels in conventional group (48.81+/-8.74) ng/L and (26.64+/-6.27) ng/L, respectively, the perindopril group was lower than the conventional group descending rates were 72.7% and 45.3% respectively.(3)The level of plasma sDR5 in CHF patients resulting from hypertensive cardiopathy was much higher than that in CHF patients resulting from any other etiological factors. CONCLUSION: DR5 may play an important role in the occurrence and progress of myocardium apoptosis of CHF patients. Perindopril can down-regulate the level of plasma sDR5, therefore, it may have the great effect on retarding course of ventricular remodeling, protecting and improving cardial function of CHF patients.

[Characterization of neutralizing activity of murine monoclonal antibodies for construction of anti-human TNF chimeric antibody].

AIM: To screen high quality murine monoclonal antibodies (mAb) for construction of anti-human TNF chimeric antibody. METHODS: 3 hybridoma cells D2, E6 and F6 were selected from one group of hybridoma cells secreting anti-human TNF mAbs and then ascitic fluids were prepared according to routine protocol. After salted out with saturated ammonium sulphate, the mAb (IgG2b) secreted by D2 cells was purified through protein A affinity chromatography column. The mAbs (IgG1) secreted by E6 and F6 cells were purified through QFF anion exchange chromatography column. The titers of 3 mAbs were detected by indirect ELISA before and after purification. Inhibition of TNF induced cell death of L929 cells and ICAM-1 upregulation on ECV304 cells were assayed as indexes of neutralizing activity of the mAbs. RESULTS: ELISA results showed that the titers of 3 mAbs D2, E6, and F6 before purification were 1x10(-6 ), 1x10(-7 ), and 1x10(-7 ), respectively, and 0.01, 0.002 and 0.002 mg/L after purification, respectively. 0.16 mg/L D2, 0.40 mg/L E6, or 0.50 mg/L F6 could protect 50% L929 cells from cell death induced by 2x10(4) U/L TNF, and the inhibition rates of ICAM-1 upregulation by 2x10(5) U/L TNF of 1.0 mg/L D2, E6 or F6 were 70.1%, 60.1%, and 60.1%, respectively. The inhibition rates still reached 60.1%,53.7% and 59.0%, respectively, when the concentrations of 3 mAbs D2, E6 and F6 decreased to 0.1 mg/L. CONCLUSION: 3 mAbs with high titers and neutralizing activities were obtained, which paves the way for construction of anti-human TNF chimeric antibody.