OBJECTIVETo establish a method for efficient induction and expansion of Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) in vitro and evaluate the possibility of using this strategy for treatment of nasopharyngeal carcinoma (NPC).

METHODSEBV-transformed B lymphoblastoid cells (BLCLs) were used as the antigen stimuli and antigen-presenting cells. EBV-specific CTL was induced by co-culture of the autologous peripheral blood mononuclear cells (PBMCs) and the irradiated BLCLs, and expanded with a cocktail method consisting of OKT-3, irradiated homologous PBMC, and IL-2. The specific activity of the CTL against the NPC cells was measured with MTT assay.

RESULTSEBV-specific CTL was successfully induced and expanded by 600 folds. The killing efficiency of the CTL was 76% for autologous BLCLs, 13% for homologous BLCLs, 51% for autologous NPC cells, and 27% for homologous CNE cell line, and after expansion, the corresponding killing efficiencies were 63%, 25%, 49%, and 33%, respectively. The non-specific killing only slightly increased after the expansion.

CONCLUSIONEBV-specific CTL can be successfully induced and expanded in vitro for specific killing of autologous NPC cells, suggesting the potential of this strategy in the treatment of NPC.

Antigen-Presenting Cells ; cytology ; immunology ; Antigens, Viral ; immunology ; B-Lymphocytes ; cytology ; immunology ; virology ; Cells, Cultured ; Coculture Techniques ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; virology ; Tumor Cells, Cultured

OBJECTIVE: To detect the expression of miR-99a-5p in myelodysplastic syndrome (MDS), to predict the target genes and to analyze its function by using bioinformatics. METHODS: The expression levels of bone marrow miR-99a-5p in MDS patients were detected by qRT-PCR, and the correlation of miR-99a-5p expression with clinical pathological characteristics, percentage of marrow blasts , chromosome karyotype and peripheral blood hemogram were analyzed. The target genes of miR-99a-5p were predicted by Targetscan, Miranda and Microcosm, and the intersection of the predicted results of 3 softwares was used as a potential target gene for miR-99a-5p. RESULTS: The expression level of bone marrow miR-99a-5p in MDS patients was significantly higher than that of healthy controls (P<0.01); According to the prognostic score of IPSS, MDS patients were divided into relatively low risk group (including low risk group and intermediate risk group 1) and relatively high risk group (including intermediate risk group 2 and high risk group). Analysis showed that the expression level of bone marrow miR-99a-5p in relatively low risk group was 2.40 times higher than that in healthy control group (P<0.01), the expression level of bone marrow miR-99a-5p in relatively high risk group was 6.66 times higher than that in healthy control group (P<0.01), the expression level of miR-99a-5p in relatively high risk group was 2.80 times higher than that in the relatively low risk group ( P<0.01) ; the expression level of bone marrow miR-99a-5p in t-MDS group was 6.35 times higher than that in healthy control group (P<0.001). There was a significant positive correlation between the expression level of miR-99a-5p and the percentage of marrow blasts (P<0.01), but the expression of miR-99a-5p did not correlate with chromosome karyotype and peripheral blood hemogram; In this study it was obtained that ST5 might participate in pathological mechanism of MDS by bioinformatic analyses and references. CONCLUSION The expression levels of miR-99a-5p is up-regulated in MDS patients, and its expression showed a rising tendency which may be involved in the pathogenesis of MDS by regulating target gene ST5.
Bone Marrow ; Computational Biology ; Humans ; Karyotyping ; MicroRNAs ; Myelodysplastic Syndromes

OBJECTIVETo investigate the expression of miR-550a-5p in bone marrow of patients with myelodysplastic syndrome (MDS), and to predict its target genes and function by bioinformatics analyses, so as to provide the evidence to furthre explore the role of miR-550a-5p and its target genes in pathogenesis of MDS.

METHODSReal-time PCR was used to detect the expression of miR-550a-5p in 54 MDS patients, 16 acute myeloid leukemia transformed from MDS (sfAML) and 19 healthy controls, and the correlation between the expression of miR-550a-5p and clinical pathologic characteristics of MDS, including chromosome, percentage of marrow blasts, absolute neutrophil count, platelet count and hemoglobin levels were analyzed. The sequence of miR-550 was searched in miRBase database. Target genes of miR-550a-5p were predicted by Microcosm,Miranda and Targetscan, and the predective results were collected, then the enrichment analyses of target gene function(GO) and signalling pathway(pathway of miR-550a-5p) were carried out by using gene ontology darabase and KEGG database.

RESULTSThe expression of miR-550a-5p in bone marrow of all MDS patients was higher than that in controls: the expression level of miR-550a-5p in low risk MDS and middl risk 1 MDS was 1.7 times of controls (P=1.23×10); the expression of miR-550a-5p in midde risk 2 MDS and high risk MDS was 1.9 times of controls (P=1.20×10); the expression of miR-550a-5p in tAML was 2.0 times of controls (P=5.61×10). The miR-550a-5p expression level was up-regulated gradually with the enhancement of disease risk of MDS, but there was no correlation between the expression level of miR-550a-5p and clinical pathologic characteristics of MDS(chromosome: Normal: 1.11±0.19, Abnormal:1.26±0.15, P>0.05; Percentage of Marrow Blasts: r=0.29,P=0.07; absolute neutrophil count: r=-0.02,P=0.89; hemoglobin level: r=0.09,P=0.57; platelet count: r=0.25,P=0.08). The sequence of miR-550 was conservative among different species, and the prediced results indicated that there were 19 target genes in intersection. The functions of target genes were enriched in regulation of stress-activated cascade, MAPK pathway, regulation of muscle organ development, regulation of protein homodimerization activity and other biological processes; they participated in some molecular functions including enzyme activity, combination processes of some molecules as protein, cAMP and domain existed in cell junction, synapse, coated vesicle, dendrite and other cellular components. Two of them-PDLIM2 and PSME1 were selected which might play a role in pathologic mechanism of MDS regulated by miR-550a-5p.

CONCLUSIONThe expression of miR-550a-5p in bone marrow of MDS patients increases specifically, and miR-550a-5p may play a role in the pathogenesis of MDS through regulation of target genes, PDLIM2 and PSME1.