Total RNA from JM cell lines secreting T leukmia derived suppressor factor was isolated using single—step method of acid guanidium followed by oligo—d(T)—cellulose affinity chromotogra-phy for purification of mRNA.TLSF_(JM)mRNA was mieroinjected into Xenopus Laevis Oocytes.The inhibitory activity in Xenopus Laevis Oocytes culture supernatant or lysate was detected by PHA plus IL—2 induced—mouse thymocyte proliferation assay The results showed that Xeno-pus Laevis Oocytes could efficiently translate exogenous mRNA,the trans ated products mainly existed inside oocytes and approximately2% of the products was secreted into the culture super-natant.Success in TLSF_(JM)mRNA translation in vitro might provide the basis for establishment of cDNA library and TLSF_(JM)gene cloning and expression.

Murine thymocytes can be induced to proliferate and differentiate into cytotoxic effector cells when stimulated with Con A and provided with various cytokines. We have analysed the requirements for a range of cytokines in this system and, i n particular, the role of transforming growth factor ? (TGF-P) in regulating the cellular response to optimal concentration of the cytokines in combination. It was found that Con A+IL-2 (?)ed to marked proliferation and t hi s was augmented with IL-6, TGF-? inhibited the prolifera tion induced by IL-2+IL-6, the presence of TGF-?from the beginning of culture inhibited the development of cytotoxic effector cells and such effects were dose-related and could be reversed with antiserum to TGF-? but not. by a range of cytoki nes including IL-2, IL-4, IL-5, IL-6, TNF-a or ?-IFN. Phenotypic analysis indicated that TGF-? preferentially inhibited the expression of IL-2 receptor and the growth of ly2~+ cells.

Biodistribution and radioimmunoimaging of monoclonal antibody HB8759 agaist human melanoma in tumor-bearing nudemice were investigated.The results showed that mean tumor: nontumor(5 main organs)ratios were 6.6 and 9.6 at 48 and 72hr after injection of the ~(131)Ⅰ—McAb,respectively,and tumor was clearly visualized under ECF at the same time.It is suggested that McAb HB8759 is a potential biologic product for diagnosis of metastic melanoma and intraocularmelanoma and clinical treatment of melanoma.

Based on the high level of human granulocyte—macrophage colony—stimulating factor(HGM—CSF)re-ceptor of HL60 cell line upon induction by DMSO,we developed a hioassay method for HGM—CSF with ahigher specificity and sensitivity.The sensitivity of this assay reached to 0.3ng/ml by selecting the optimalHL60 cell—induced time,cell density and GM—CSF—stimulated time.With this assay,we are able to quantifythe level of rHuGM—CSF as well as conditioned media from human bladder carcinoma cell line U5637 screatinga high level of GM—CSF.

Although adhesion molecules have long been recognized to be differentially expressed on naive and effector/memory T cells,it has recently been found that a number of chemokine receptors are also differentially expressed on T cells,depending on their Ag experience and type of polarization. Recent data suggests that chemokines and their receptors are essential elements that regulate the positioning of T cells and their partners for priming and T helper 1 (Th1)- or Th2-mediated responses,therefore,are probably the most promising targets for treating immune diseases.

Objective:To identify the function of the 5′-flank upstream regulation region of human CD226 gene.Methods:The upstream regulation region of CD226 was cloned by PCR and ligated into pGL3 vector. Then the vector was transfected into Jurkat cell and luciferase activity was detected after 48 h culture.Results:CD226 gene may have two promoters, P1 and P2,which were located at the region of -843--319 bp and +1-+181 bp respectively, and PMA can up-regulate P1 while down-regulate P2. Both P1 and P2 can be up-regulated by A23187, especially P2.Conclusion:CD226 gene may have two promoters, and PMA and A23187 can regulate CD226 promoter activity in the similar pattern of protein level.

BACKGROUND: Biological function of leukocyte-associated immunoglobulin like-receptor 1 (LAIR 1) has clearly researched in China and abroad, but the in vivo biological function of LAIR is poorly understood. OBJECTIVE: To establish LAIR-2 (CD306) eukaryotic expression vectors and to purify and identify the fusion protein. DESIGN, TIME AND SETTING: A single sample observation experiment was performed at the Fourth Military Medical University of Chinese PLA between June 2007 and June 2008.MATERIALS: plg/3c vector was offered by Oxford University, pcDNA3.1 vector was provided by Meyaard doctor. Chinese hamster ovary (CHO) cell lines were preserved by the Department of Immunology, Fourth Military Medical University of Chinese PLA.METHODS: Two eukaryotic expression vectors plg/3c-LAIR-2 and pcDNA3.1-LAIR-2 were constructed and were transfected into CHO cells. The binding activities of LAIR-2 fusion protein to LAIR-2 mAbs were identified by Western blot, immunocytochemistry and flow cytometry assay.MAIN OUTCOME MEASURES: The construction of stably transfected cell lines, and the purification and identification of fusion protein. The activity of LAIR 2 protein combined to corresponding monoclonal antibody.RESULTS: Eukaryotic expression vectors were constructed and trasnsfected into CHO cells successfully. Two cells lines CHO/LAIR-2-Fc and CHO/LAIR-2 that steadily expressed LAIR-2-Fc fusion protein and LAIR-2 protein were established. Western blot assay showed that LAIR-2 protein could bind specially to LAIR-2 mAb 1A7, 3H12 and 4A9. Immunocytochemistry and flow cytometry assay demonstrated that 3H12 and 4A9 could bind to LAIR-2 expressed in the transfected CHO cells. CONCLUSION: Two ceils lines CHO/LAIR-2-Fc and CHO/LAIR-2 were successfully constructed, which can transfected to CHO cells. The eukaryotic expressed LAIR-2 protein has good binding activity to LAIR-2 mAbs.

Objective:To investigate the expression and function of CD226 on NK s ubsets and its coexpression with other activation receptor and inhibition recept or on NK cells. Methods:The expression of CD226 on CD56 bright and CD56 dim NK subsets and coe xpress ion with CD16 and NKG2A in PBMC and MLC in the presence or absence of IL-2 or IL-15 were detected by double fluorescent staining and flow cytometry analysis. The level of IFN-? in the supernatants of PBMC culture and MLC treated with o r without IL-2 or IL-15 were evaluated by ELISA. 51Cr release assay w as employed to measure the specific lysis of NK cells killing target K562 cells. Results:CD226 was mainly expressed on CD56 dim NK subsets in PBMC. When s imulated by IL-2, CD226 expression was shifted to CD56 bright NK subsets, while IL-15 increased the subpopulation of NKG2A+CD226+double positive cell s. In MLC-generated NK cells, CD226 was mainly expressed on CD56 dim NK su bsets, and also shifted to CD56 bright NK subsets in the addition of IL-15 . Furt hermore, the percentage of CD16+CD226+和NKG2A+CD226+ subsets were increa sed when stimulated by IL-2 or IL-15. There was great increase in IFN-? lev el in the supernatants of PBMC culture in the presence of IL-2 or IL-15, but no difference in the supernatants of MLC treated with or without two cytokine s. Moreover, the cytotoxicity of NK cells in PBMC and MLC were greatly enhanced by IL-2 or IL-15. Conclusion:CD226 is mainly expressed on CD56 bright NK subsets in IL-2 or IL-1 5 activated NK cells, and is coexpressed with CD16 and NKG2A preferentiatly, whi ch maybe involve in the modulation of cytotoxicity of NK cells based on the balance of coexpressed activation and inhibition receptors. [

Objective:To investigate the impact of alanyl-glutamine(Ala-Gln) on outcome in radiation enteritis rats.Methods: Male SD rats(n=70)were separated randomly into four groups: control group(n=10),AR+pseudosurgery group(n=20),AR+TPN group(n=20) and AR+TPN+Ala-Gln group(n=20).Rats were observed for mortality,changs of body weight,villous hight and area,the bcteriral translocation(BT)in mesenteric lymph nodes(MLNs),liver,spleen and peritoneal cavity.Serum TNF-? and sIL-2R level were determined by sandwich ELISA.Results: When Ala-Gln was administered in radiation enteritis rat,the mortality,body weight loss and bacterial translocation were decreaded,the villous hight and area was increased and the TNF-? and sIL-2R levels were reduced.Conclusion: Parenteral Ala-Gln nutrition can improves the results of radiation enteritis rats.

Objective: To construct and express the eukaryotic expression vector of human pSecTag2B-CD226(PTA1).Methods: The gene fragment encoding extracellular region of human CD226 was cloned into the eukaryotic expression vector pSecTag2B.After sequencing,the vector was transfected into COS-7 cells,and the expressed molecule was purified by affinity chromatography.Finally,the product was characterized by ELISA.Results: hCD226-6His was successfully expressed.After purification,the concentration of hCD226-6His was 50?g/ml.Conclusion: Human CD226-6His fusion protein involving the extracellular region of CD226cDNA has been successfully expressed and purified,which helps prepare the ground for further functional studies of this molecule.