Guo-Rong Luo

Serum immunoreactivity of SMP30 and its tissues expression in hepatocellular carcinoma

OBJECTIVES To detect serum antibody against SMP30 in HCC patients and to evaluate its potential associations with HCC patients clinical parameter and expression levels in HCC tissues. DESIGN AND METHODS Serum antibody to SMP30 was tested by ELISA method; SMP30 mRNA and protein expression in HCC patients were analyzed using the methods of in situ nucleic acid hybridization and immunohistochemistry, respectively. RESULTS The highest relevance of SMP30 antibody was associated with HCC (32.4%). The positive rate of SMP30 antibody was not related to the age of patients, tumor size, metastasis and infections of HBV, but the positive rate for SMP30 antibody in the HCC sera with alpha-fetoprotein (AFP) negative was higher (43.6%) compared with that AFP positive (26.2%). Both SMP30 mRNA and protein expression levels were downregulated in HCC and upregulated in adjacent tissues. CONCLUSIONS SMP30 may be useful for HCC serologic screening, especially for the patients with AFP negative.

Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+ CD28+ T lymphocyte responses

OBJECTIVE To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8(+) CD28(+) cytotoxic T lymphocyte (CTL) responses. METHODS Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2K(b): Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8(+)CD28(+) CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. RESULTS Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8(+)CD28(+)CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- γ under the stimulation of H-2K(b): Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells. CONCLUSIONS The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8(+) CD28(+) CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

Knockdown of GCF2/LRRFIP1 by RNAi Causes Cell Growth Inhibition and Increased Apoptosis in Human Hepatoma HepG2 Cells

BACKGROUND GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. MATERIALS AND METHODS GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. RESULTS Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. CONCLUSIONS These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

A constructed HLA-A2-restricted pMAGE-A1278–286 tetramer detects specific cytotoxic T lymphocytes in tumour tissues in situ

Objective To construct a human leucocyte antigen (HLA)-A2-restricted peptide 278–286 of melanoma-associated antigen family A, 1 (pMAGE-A1278–286) tetramer to analyse the distribution of cytotoxic T lymphocytes (CTLs) in tumour tissue and tumour-adjacent normal tissue. Methods A HLA-A2-pMAGE-A1278–286 tetramer was constructed. The distribution of pMAGE-A1278–286-specific CTLs was investigated in tumour tissues and tumour-adjacent normal tissues from patients with hepatocellular carcinoma using in situ HLA-A2-pMAGE-A1278–286 tetramer staining. Results Sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis indicated that HLA-A2 heavy and light chain proteins were successfully obtained. The successful construction of the HLA-A2-pMAGE-A1278–286 monomer was confirmed with Western blot analysis using W6/32 antibody. Flow cytometry confirmed the specific binding of HLA-A2-pMAGE-A1278–286 tetramer to pMAGE-A1278–286-specific CTLs. In situ HLA-A2-pMAGE-A1278–286 tetramer staining demonstrated that the number of pMAGE-A1278–286-specific CTLs in tumour tissues was significantly higher than in tumour-adjacent normal tissues. Conclusions The HLA-A2-pMAGE-A1278–286 tetramer was useful for the detection of pMAGE-A1278–286-specific CTLs in both tumour tissues and tumour-adjacent normal tissues. In situ tetramer staining is a powerful tool for investigating the distribution of pMAGE-A1278–286-specific CTLs in the tumour microenvironment.

Fusion of EGFP and porcine α 1,3GT genes decrease GFP expression

Abstract Objective To investigate the effect of fusion proteins expressed by the fused gene of porcine α 1, 3 galactosyltransferase (α1, 3 GT) and enhanced green fluorescent protein (EGFP) on the green fluorescence intensity of EGFP. Methods The fragment containing α 1, 3GT was firstly recovered after the pcDNA3.1- α 1, 3GT recombinant vector were digested with Bam HI and Eco RI, and then, the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes. The new recombinant eukaryotic expression pEGFP/α 1, 3GT vector was obtained and sequenced. The pEGFP/α 1, 3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT, and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry. Results The positive percentage of A549 was 80.5%, and that of 293 FT was 86.5% 48 hours after the two cell lines both were transfected by pEGFP-N1. The positive percentage of A549 was 75.8%, and that of 293 FT was 81.2% 48 hours after the two cell lines were transfected by pEGFP/α 1, 3GT. The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 1.21 and 0.956, respectively when compared with that of A549 without transfection. Meanwhile, the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 7.66 and 1.00, respectively when compared with that of 293FT cells without transfection. Conclusions These results suggested that the expression of EGFP gene fused with porcine α 1, 3GT gene was partly inhibited.

Serum immunoreactivity of cancer/testis antigen OY-TES-1 and its tissues expression in glioma

OY-TES-1 is a member of the cancer/testis antigen family that is expressed in healthy testis tissue and certain types of cancerous tissue. The present study aimed to analyze the expression pattern of OY-TES-1 and serum anti-OY-TES-1 antibody concentration in patients with glioma. OY-TES-1 mRNA was detected in 28/36 (78%) of glioma cases using conventional reverse transcription polymerase chain reaction (RT-PCR) analysis. RT-quantitative-PCR revealed that OY-TES-1 was expressed at a higher level in glioma tissues compared with normal adult tissues (with the exception of testis tissue). Anti-OY-TES-1 antibodies were present in the serum of 5/36 (14%) of patients with glioma, but absent in all the serum samples from 107 healthy donors. Immunohistochemical analysis demonstrated that OY-TES-1 protein was expressed in all glioma tissues from patients with anti-OY-TES-1 antibody seropositivity. These results suggest that OY-TES-1 is a novel candidate for glioma immunotherapy.