To evaluate the efficiency of three in vitro refolding methods for a humanized single-chain Fv antibody against human CTLA4(CD152) expressed in E. coli, the denatured and purified inclusion bodies (IBS) were refolded by dilution, dialysis and in situ refolding via Immobilized Metal-Ion-Affinity Chromatography (IMAC), respectively. The concentration of refolded scFvs was examined by Bradford method. And the antigen binding activity of the refolded scFvs was analyzed by indirect cell-ELISA. The highest and lowest refolding yields could be obtained by dialysis and in situ refolding via IMAC, respectively. The binding activity of the refolded scFv by dialysis was 1.95-fold higher than that by dilution, 4.13-fold higher than that by in situ refolding via IMAC (GSH/GSSH excluded) and 3.63-fold higher than that by in situ refolding via IMAC (GSH/GSSH included), respectively. In conclusion, a high refolding yield and binding activity of scFv with natural conformation could be obtained by dialysis in the condition of 0. 15 mol/L sodium chloride, 50 mmol/L Tirs-HCl, pH 8. 0 buffer containing 3 mmol/L reduced glutathione and 1 mmol/L oxidized glutathione for 48 hours at 4 degrees C.
Antigens, CD ; biosynthesis ; genetics ; immunology ; Antigens, Differentiation ; biosynthesis ; genetics ; immunology ; CTLA-4 Antigen ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

The expression of inducible co-stimulator (ICOS) in peripheral blood T lymphocytes from the patients with systemic lupus erythematosus (SLE) and the role in the pathogenesis of SLE was investigated. By using two-color immunofluorescent staining and flow cytometric assay, the expression levels of ICOS in peripheal blood T lymphocytes from 33 patients with SLE and 16 healthy volunteers were detected. SLE diseases activity index (SLEDAI) of the patients with SLE was used to evaluate the disease activity. The correlation between the ICOS expression and SLEDAI was analyzed among the groups. The results showed that the expression levels of ICOS in T lymphocytes in active SLE group was markedly higher than those in the control and inactive SLE groups (both P< 0.01). There was no significant difference in the expression levels of ICOS between the inactive SLE and the control groups (P>0.05). In active SLE and inactive SLE groups, positive linear correlation was found between the levels of the ICOS expression in T lymphocytes and SLEDAI (r=0. 711, P=0.001; r=0.561, P=0.03). It was suggested that the expression of ICOS in peripheral blood T lymphocytes from the patients with active SLE was up-regulated and and ICOS might be related to the pathogenesis of SLE.
Antigens, Differentiation, T-Lymphocyte/*biosynthesis ; Antigens, Differentiation, T-Lymphocyte/genetics ; Lupus Erythematosus, Systemic/blood ; Lupus Erythematosus, Systemic/etiology ; Lupus Erythematosus, Systemic/*immunology ; T-Lymphocytes/*immunology

The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.
Anti-Infective Agents ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; genetics ; Cyanogen Bromide ; pharmacology ; Escherichia coli ; genetics ; metabolism ; GTP-Binding Protein alpha Subunits, G12-G13 ; biosynthesis ; genetics ; Inclusion Bodies ; metabolism ; Protein Structure, Tertiary ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thioredoxins ; genetics ; Transfection

OBJECTIVE: To evaluate the expression of hCTLA4-Ig and their biological function in newborn porcine islets (NPIs) transfected with AAV-hCTLA4-Ig. METHODS: Cultured NPIs were transfected with AAV-hCTLA4-Ig. The expression of CTLA4-Ig in these NPIs was assayed by RT-PCR and immunocytochemistry. The levels of IL-2, IFN-gamma, and TNF-alpha in the culture medium were assayed by ELISA after these cells the co-cultured with human. The response of glucose-stimulated insulin secretion was observed in the transgene group and the control group. RESULTS: The expressions of CTLA4-Ig mRNA and protein were detected in the transgene group. The levels of cytokines were obviously lower in the transgene group than those in the control group (P<0.01). There was no significant difference in the response of glucose-stimulated insulin release between the transgene group and the control group (P>0.05). CONCLUSION AAV mediated hCTLA4-Ig expression in NPIs could inhibit T lymphocyte to produce cytokines, while the endocrine functions of the NPIs were not significantly affected.
Animals ; Animals, Newborn ; Antigens, CD ; biosynthesis ; genetics ; Antigens, Differentiation ; biosynthesis ; genetics ; CTLA-4 Antigen ; Cells, Cultured ; Dependovirus ; genetics ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunohistochemistry ; Interferon-gamma ; analysis ; Interleukin-2 ; analysis ; Islets of Langerhans ; cytology ; immunology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Transfection ; Tumor Necrosis Factor-alpha ; analysis

The role of CD80/CD86 and CTLA-4 in the pathogenesis of systemic lupus erythematosus and their clinical significance was investigated. By using RT-PCR technique, the expression of CD80/CD86 and CTLA-4 mRNA in peripheral blood mononuclear cells (PBMC) were semiquantitatively detected in 32 patients with active SLE. The results showed that the percentage of positive CD86 expression in active SLE was increased significantly as compared with normal controls (90.63% vs 60.00%, P<0.01). The mean level of CD86 mRNA expression in active SLE group was markedly higher than in the normal controls (0.6410+0.0174 vs 0.4510+0.0402, P<0.001). Compared with normal controls, the percentage of positive CTLA-4 expression and the mean level of CTLA-4 mRNA expression in active SLE group were both increased significantly (both P<0.01). There was no statistical differences in positive rate of CD80 and the average level of CD80 mRNA between the two groups (both P>0.05). It was concluded that the aberrant expression of CD86 and CTLA-4 might play an important role in the activity and development of SLE.
Adolescent ; Adult ; Antigens, CD ; Antigens, Differentiation ; biosynthesis ; genetics ; B7-1 Antigen ; biosynthesis ; genetics ; B7-2 Antigen ; biosynthesis ; genetics ; CTLA-4 Antigen ; Female ; Flow Cytometry ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lupus Erythematosus, Systemic ; blood ; etiology ; Lymphocyte Activation ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.
Adipogenesis/drug effects ; Alkaline Phosphatase/biosynthesis/genetics ; Antigens, Differentiation/biosynthesis/genetics ; Bone Marrow/pathology ; Cell Differentiation/drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/biosynthesis/genetics ; Culture Media ; Gene Expression Regulation ; Humans ; Lipoprotein Lipase/biosynthesis/genetics ; Mesenchymal Stem Cells/*drug effects/metabolism/pathology ; Osteoblasts/drug effects/*metabolism/pathology ; Osteogenesis/drug effects ; PPAR gamma/biosynthesis/genetics ; Parathyroid Hormone/*pharmacology ; Peptide Fragments/*pharmacology ; Receptor, Parathyroid Hormone, Type 1/antagonists & inhibitors

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; genetics ; Cytarabine ; pharmacology ; Erythrocytes ; cytology ; metabolism ; ultrastructure ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Macrophages ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Time Factors

OBJECTIVE: To create a method for transfecting human vascular endothelial growth factor165 (hVEGF165) gene into bone marrow mesenchymal stem cells (MSCs) in rats. METHODS: MSCs of Wistar rats were isolated by density gradient centrifugation and purified based on their ability of adhesion to plastic. Detections of cell surface antigens, including CD34, CD45, CD44, and SH3, were performed using flow cytometry. MSCs' potential of differentiating into osteoblast and lipoblast in vitro was tested. The vector pcDNA(3.1)-hVEGF165 was transfected into MSCs with the liposome mediated method. The expression of hVEGF165 in the transfected cells was detected by enzyme linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: The cultured MSCs were CD34-, CD45-, CD44+ , and SH+, which were differentiated into osteoblasts and lipocytes successfully. The expressed hVEGF165 in the transfected rat MSCs was demonstrated. CONCLUSION The vector pcDNA(3.1)-hVEGF165 is successfully expressed in MSCs.
Animals ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Humans ; Hyaluronan Receptors ; analysis ; Leukocyte Common Antigens ; analysis ; Mesenchymal Stem Cells ; cytology ; metabolism ; Rats ; Rats, Wistar ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo investigate the relationship between the expression of host human leukocyte antigen-B mRNA (HLA-B mRNA) and HLA-B antigen in peripheral blood leukocytes (PBLs) and the differentiation and metastasis of gastric carcinoma (GC).

METHODSTo design and screen specific primers of HLA-B gene independently, detect the expression of HLA-B mRNA from 30 GC patients by reverse transcription-PCR and compare with the HLA-B antigen expression measured by flow cytometry.

RESULTSThe expression rate of PBL HLA-B mRNA from GC patients (23. 3% ) was very significantly lower than that of normals (87. 5% ) (P <0. 01) , especially concerning the poorly differentiated GC patients with lymph node metastasis (16. 0% ). Measured by flow cytometry, the expression percentage of HLA-B antigen of well-differentiated GC patients without lymph node metastasis was 88. 2% , an obviously decreasing tendency was showed in comparison with that in the normal group (98. 8% ) , although the difference was not significant (P = 0. 056) , and the expression percentage in poorly differentiated GC patients with lymph node metastasis(73. 3% )was declined significantly (P <0. 05).

CONCLUSIONThe expression of PBL HLA-B mRNA and HLA-B antigen in GC patients is decreased or lost, and correlated with differentiation and metastasis of the cancer. The expression of PBL HLA-B mRNA may more directly reflect its relationship with the tumor differentiation and metastasis than that of HLA-B antigen.

Adult ; Aged ; Cell Differentiation ; Female ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; HLA-B Antigens ; analysis ; genetics ; Humans ; Leukocytes ; immunology ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms ; genetics ; immunology ; pathology

Recent studies revealed that apoptotic cells are actively involved in immunosuppression and anti-inflammation. After being phagocytosed by macrophages, apoptotic cells can actively regulate cytokines secretion from lipopolysaccharide (LPS)-stimulated macrophages, in which the secretion of immunosuppressive cytokines such as interleukin-10 (IL-10) is increased while the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFa), interleukin-1beta (IL-1b) and leukin-8 (IL-8) are suppressed. In this paper, we first present evidence that phagocytosed apoptotic cells regulate cytokine secretion of LPS-stimulated macrophages, but also inhibit the activation of T lymphocytes stimulated by ConA. These data suggest that apoptotic cells can alter the biological behavior of macrophages which gain immunosuppressive property.
Animals ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Apoptosis ; immunology ; Chemokine CXCL2 ; Chemokines ; biosynthesis ; genetics ; Concanavalin A ; pharmacology ; Cytokines ; biosynthesis ; Female ; Humans ; Immune Tolerance ; In Vitro Techniques ; Jurkat Cells ; Lectins, C-Type ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; drug effects ; Macrophages ; drug effects ; immunology ; Mice ; Mice, Inbred ICR ; Phagocytosis ; Receptors, Interleukin-2 ; metabolism ; Signal Transduction ; immunology ; T-Lymphocytes ; drug effects ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics