Construction and screening of human immunodeficiency virus-1 negative regulation factor peptide-specific CD4+T lymphocyte clone / 中华传染病杂志

Objective To construct and screen the human immunodeficiency virus-1 (HIV-1) negative regulation factor (Nef) peptide-specific CD4+ T lymphocyte clone.Methods Peripheral blood mononuclear cells (PBMC) from five asymptomatic HIV-1 infected patients were collected and Bulkcultured with Nef end peptides.The CD4 molecule and intracellular interferon (IFN)-gamma of cultured cells were detected by two-color flow cytometry.The Nef end peptide-specific T cell clone was then constructed by limited dilution and confirmed through enzyme linked immunospot assay (ELISPOT).The best grown cells were selected and cultured as the final clone.Results The Nef end peptide-specific-T lymphocyte clone was successfully constructed from PBMC of one HIV-infected patient and confirmed by ELISPOT.The detection of human leukocyte antigen (HLA)-DRB1 type showed that the epitope of this peptide was probably HLA-DRB1 * 0406.Conclusion The Nef end specific-T cell clone is successfully constructed,and a new epitope in the C-terminus of Nef protein and its HLA restriction are identified.

Detection of Putative T cell Clones Using T cell Receptor beta Chain Gene Clonality Assay in Korean Patients with Aplastic Anemia / 대한진단검사의학회지

BACKGROUND: We analyzed T cell receptor beta chain (TCRB) gene to investigate the presence of putative T cell clones and its clinicopathologic implications in Korean patients with aplastic anemia (AA). METHODS: Twenty-nine bone marrow specimens were collected from 20 AA patients, 19 specimens from initial diagnosis and 10 from follow-up. T cell clonality assay was performed using IdentiClone(TM) TCRB Gene Clonality Assay kit (InVivoScirbe Technology, USA) and automatic genetic analyzer. Patients' clinical information and laboratory parameters were also analyzed. RESULTS: Five patients had definitive underlying factors related with aplastic anemia, such as hepatitis B virus (4 cases) and benzene exposure (1 case). Putative T cell clones were detected in bone marrow specimens of 11 (58%) out of 19 patients at diagnosis. The location of putative T cell clones of TCRB gene (diversity region, Dbeta; joining region, Jbeta; variable region, Vbeta) was distributed in Dbeta2+Jbeta2 (6 cases), Dbeta1+Jbeta1 (3 cases), Vbeta+Jbeta1 (2 cases), and Dbeta1+Jbeta2 (2 cases). Interestingly, among seven patients who underwent stem cell transplantation, five patients with no T cell clones detected at diagnosis developed new T cell clones during the follow-up. CONCLUSIONS: Putative pathogenetic T cell clones were detected in most of AA patients in the current study. T cell clonality assay would be useful for investigating the pathophysiology of acquired AA.

Clonal Heterogeneity of Human Bone Marrow-Derived Stromal Cells / 대한정형외과연구학회지

PURPOSE: The isolated human bone marrow-derived stromal cells (BMSCs) were expanded and showed multipotentiality for differentiation to both adipocytes and osteoblasts. We selected the multiple clones and characterized their proliferation potentials, surface markers and differentiation potentials. MATERIALS AND METHODS: BMSCs were isolated from bone marrow. After forming colonies, colonies were selected and cultured. We performed MTT assay and FACS. Cultured colonies were differentiated into adipocyte and osteoblast. RESULTS: BMSCs were isolated and plated in tissue-culture dishes. The number of clones was about 30. Clones were divided into three groups. Group I was round and small in size, and showed rapid proliferation. Group II showed similar pattern in cell size and proliferation with group I clones during early passage. But, after 4 passages, the clones became flat and larger cells and showed slower proliferation. Finally, group III clones were flat and large cells and replicated slowly in early passage. Group I was fibroblastic in morphology, but Group II changed to flat and larger cells after passage 4. FACS and proliferation assay were performed at passage 5. Group I and Group II cells differed in their expression of the cell-surface epitopes, CD29 and CD105. Group I cells were differentiated into adipocytes better than Group II cells. CONCLUSION: In this study, these results indicated that cell surface markers were expressed differently for clones having different differentiation properties. These clonal BMSCs may also be used for the identification of lineage-determining factors.

Mutation of DNA Mismatch Repair Genes and its Relation to Taxol and Topotecan Chemosensitivity in the Clones from the Cisplatin-Resistant Ovarian Cancer Cell Lines / 대한산부인과학회잡지

OBJECTIVE: Ovarian cancer represents a relatively chemosensitive solid tumor, with responsiveness to a range of agents. Cisplatin is the mainstay of drug treatment and is one of the most active single agent. However, the overall outcome for patients remains unsatisfactory and the emergence of drug resistance is a major factor in treatment failure. Loss of DNA mismatch repair is a common finding in many types of sporadic cancer as well as in patients with hereditary nonpolyposis colon cancer. Cells that lack DNA mismatch repair are resistant to commonly used chemotherapeutic agents. Selection of cells for resistance to cisplatin, a well-recognized mutagen, could result in mutation in genes involved in DNA mismatch repair. METHODS: This study evaluated the mutation of hMLH1 and hMSH2, and its relation to the Taxol and Topotecan chemosensitivity in the clones from the ovarian cancer cell line 2008 and cisplatin-resistant cell line 2008/ C13*5.25. RESULTS: 1. Cells from 2008 and 2008/C13*5.25 expressed both hMLH1 and hMSH2 when analysed with immunoblotting. 2. Twenty two out of 100 single-cell clones from 2008 and 27 of clones from 2008/C13*5.25 expressed no hMLH1. hMSH2 was expressed in all clones. 3. There was no difference of Taxol chemosensitivity between 2008 and 2008/C13*5.25 cell lines. In the 2008/C13*5.25 cell line, the hMLH1-deficient clones were more sensitive to Taxol than the hMLH1-proficient clones(P=0.049), but in 2008 cell lines hMLH1-proficient clones were more sesitive to Taxol(P=0.003). 4. There was no difference in Topotecan chemosensitivity between 2008 and 2008/C13*5.25 cell lines. In the 2008/C13*5.25 cell line, the hMLH1- deficient clones were not more sensitive to Topotecan than the hMLH1-proficient clones. In the 2008 cell lines hMLH1-deficient clones were more sesitive to Topotecan(P=0.001). Overall, hMLH1-deficient clones from both 2008 and 2008/C13*5.25 cell lines were significantly more sensitive to Topotecan(P=0.001). 5. Microsatellite instability was not demonstrated in all 4 types of single-cell clones from 2008 and 2008/C13*5.25 cell lines. CONCLUSIONS: The present results indicate that there is no relation between mutation of mismatch repair gene and cisplatin resistance. But hMLH1-deficient ovarian cancer cells are more sensitive to Taxol or Topotecan in this study. The latter finding mandates the examination to assess the mutation of hMLH1 in tumor cells before treatment or at the time clinical resistance to cisplatin develops in ovarian cancer.

Cytogenetic Studies of High Dose EGF Treated KUMA3 Cell Clone / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Epidermal growth factor (EGF), directly stimulates epidermal growth and differentiation. The combination of EGFR activation and genetic alternations may lead to neoplasia and metaplasia. To study the change of chromosomal number and the aberrations of chromosomal structure of KUMA3 cell line treated with high dose EGF. MATERIALS AND METHODS: The high dose EGF treated cell clones were obtained from KUMA3 cell line which was established from squamous cell carcinoma of the lower lip by culturing cells in medium containing high dose EGF for 6 months. The chromosomal analysis and subculture were performed at subsequent passage of 1 month interval. RESULTS: In high dose EGF treated cell clones, there was no apparent change in chromosomal number, but the ratio of the number of chromosome 7 to mode chromosome number was similar to normal value (0.043). The new chromosomal structural aberrations appeared first from 30 passage of IR-200 cell clone. The chromosomal aberrations were del(1)(q23-->qter) and del(4)t(1:4)(1qter-->1q23: : 4p16-->4qter). CONCLUSION: There was no change in chromosomal number, but the ratio of the number of chromosome 7 to mode chromosome number was similar to normal value (0.043), and the new chromosomal structural aberrations were appeared.

Establishment of a long-term cytotoxic T cell line against syngeneic mouse leukemia and its biological characteristics / 中国免疫学杂志

Cytotoxic T lymphocytes (CTL) derived from the spleen of SW-1 mice immunized in vivo with syngeneic mouse leukemia clonal cells LAC-1 were cultured in TCGF containing media and tested for cytotoxicity against LAC-1 cells in a routine ~(51)Cr-release assay. The experimental results showed that the CTL cell line was proved to be Thy 1 and Lyt 2, and therefore they were considered to be of T cell lineage. When CTL were expanded in the presence of TCGF, an augmented cytolytic activity in parallel with the increase of Thy 1 and Lyt 2 cells was shown. This cytotoxic T cell line had been maintained in a long-term culture for nearly 1 year and retained the initial reactivity. Specificity studies of the CTL showed that the cell line was highly specific for LAC-1 cells and its parent line L783 Leukemic cells and the cytolytic effect was H-2 restriction. The cytotoxicity of the CTL could be blocked by conventional or monoclonal antibodies against cell surface antigen (TSA) of LAC-1 cells. Antisera against H-2 antigens, shared by LAC-1 cells, also showed blocking activity.

CD226(PTA1) monoclonal antibody induced the cytotoxicity of NK cell clone in redirected cytotoxicity assay / 中国免疫学杂志

Objective:To investigate the expression and function of CD226 on NK cell clone Methods:NK cell clone was established by limited dilution, and identified by FCM The function of CD226 on the cytotoxicity of NK cell clone was detected by RCA and the NK cell clone secreted cytokines in the supernatants during the killing phase were measured by ELISA Results:One NK cell clone was obtained by limited dilution The cytotoxicity of this clone was upregulated markedly by CD226 mAb, and the secretion levels of IFN ? and GM CSF by NK cell clone increased obviously in RCA Conclusion:CD226 is a novel NK cell activation receptor, and the elevated IFN ? and GM CSF levels may be related to CD226 mAb enhanced NK function