OBJECTIVETo investigate the expressions of CD1a and CD83 of Langerhans cells (LC) in the lesions of epidermodysplasia verruciformis (EV) patients.

METHODSWe used immunohistochemical method to detect the expressions of CD1a and CD83 in the lesions of 10 patients with EV lesions and in the skins of 10 normal subjects.

RESULTSNo CD83 + LCs was detected in all EV patients and normal controls, but CD1a + LC was found in all cases. The quantity of CD1a + LCs in the lesions of EV patients was significantly lower than that in the normal skin (P < 0.01); furthermore, the distribution of LCs in EV lesions was uneven.

CONCLUSIONThe functions of LCs may be inhibited in EV patients.

Antigens, CD ; biosynthesis ; genetics ; Antigens, CD1 ; biosynthesis ; genetics ; Epidermodysplasia Verruciformis ; immunology ; pathology ; Humans ; Langerhans Cells ; immunology ; Leukocyte Immunoglobulin-like Receptor B1 ; Receptors, Immunologic ; biosynthesis ; genetics ; Skin ; immunology ; pathology

OBJECTIVETo explore the regulational effect of oxidized low-density lipoprotein (Ox-LDL) on expression of type A scavenger receptor (SR-A) in human mesangial cells (HMC).

METHODSHMC line (HMCL) with high expression of SR-A (HMCL-SRA) was established after stable transfection of expressive vector with cDNA encoding SR-A. Uptake of Ox-LDL by HMCL was evaluated using Oil Red "O" staining. SR-A mRNA expression was examined using reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSMore uptake of Ox-LDL was observed in the HMCL-SRA than that in the untransfected HMCL. Ox-LDL could induce SR-A mRNA expression in HMC in a dose-dependent manner, and reached a peak level after 24 h of stimulation. After 24 h of stimulation with Ox-LDL at the dose of 10, 50 and 100 micrograms/ml, SR-A mRNA expression was up-regulated to 1.35, 1.83 and 2.30-fold of controls, respectively. However, LDL had no effect on the expression of SR-A.

CONCLUSIONSIt suggests that SR-A be a major binding receptor to uptake Ox-LDL in HMC. Ox-LDL may promote the progression of chronic renal diseases through up-regulation of SR-A.

Cells, Cultured ; DNA, Complementary ; Glomerular Mesangium ; cytology ; metabolism ; Humans ; Lipoproteins, LDL ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Immunologic ; biosynthesis ; genetics ; Receptors, Scavenger ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class A ; Transfection ; Up-Regulation

OBJECTIVETo construct eukaryotic expression vectors for HA-tagged receptor for advanced glycation end products (RAGE) mutants.

METHODSSite-directed mutagenesis was applied to wild-type RAGE gene cloned in the pcDNA3 vector with HA tag to obtain the mutants pcDNA3-HA-RAGE(S391A), pcDNA3-HA-RAGE(S399A), pcDNA3-HA-RAGE(S400A), and pcDNA3-HA-RAGE(T401A). After identification by sequencing, the mutants were transfected into HEK293 cells, and the expression of these mutants were detected by Western blotting using anti-HA antibody.

RESULTSThe HA-tagged RAGE mutants constructed were verified successfully by sequencing, and highly expressed in HEK293 cells.

CONCLUSIONThe success in constructing HA-tagged RAGE mutants, which are highly expressed in eukaryotic cells, may facilitate the functional study of RAGE in cell signal transduction.

Cell Line ; Cloning, Molecular ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; biosynthesis ; genetics

In order to study how to induce tolerogenic dendritic cells in vitro and its mechanism, the K562 cells transduced with HLA-G construct were used to co-culture with DC. Then their related immunological changes, such as membrane molecules CD80, CD86, ILT3 and ILT4 expression levels were detected by flow cytometry. Allogeneic proliferation of peripheral blood mononuclear cells (PBMNC) was detected by mixed lymphocyte reaction. The results showed that CD80 and CD86 expressions on DC were downregulated, while ILT3 and ILT4 expressions were upregulated after co-culturing with K562-HLA-G cells. The DCs were less able to stimulate the allogenic PBMNC. It is concluded that the membrane-bound HLA-G can upregulate expression of inhibitory receptors ILT3 and ILT4, inducing tolerogenic DC in vitro, which may provide a novel strategy for transplant tolerance induction.
B7-1 Antigen ; biosynthesis ; B7-2 Antigen ; biosynthesis ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; HLA Antigens ; immunology ; HLA-G Antigens ; Histocompatibility Antigens Class I ; immunology ; Humans ; Immune Tolerance ; immunology ; K562 Cells ; Lymphocyte Culture Test, Mixed ; Membrane Glycoproteins ; biosynthesis ; Receptors, Cell Surface ; biosynthesis ; Receptors, Immunologic ; biosynthesis ; T-Lymphocytes ; immunology ; Transfection

OBJECTIVETo study the expression of the receptor for advanced glycation end products (RAGE) and the inhibitory effect of advanced glycation end products (AGEs) on testosterone production in rat Leydig cells.

METHODSRat Leydig cells were primarily cultured and the expression of RAGE in the Leydig cells was detected by RT-PCR and immunofluorescence staining. The Leydig cells were treated with AGEs at the concentrations of 25, 50, 100 and 200 microg/ml, respectively, and the testosterone content was determined by ELISA.

RESULTSRT-PCR and immunofluorescence staining exhibited the expression of RAGE in the rat Leydig cells. AGEs remarkably suppressed hCG-induced testosterone production in the Leydig cells in a concentration-dependent manner in the 50, 100 and 200 microg/ml groups as compared with the control (P < 0.01).

CONCLUSIONRAGE exists in rat Leydig cells and AGEs can significantly inhibit the secretion of testosterone in primarily cultured rat Leydig cells.

Animals ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Glycation End Products, Advanced ; pharmacology ; Leydig Cells ; metabolism ; radiation effects ; Male ; Rats ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Testosterone ; biosynthesis

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-gamma, TNF-alpha, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-alpha and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-gamma secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+CCR4-CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.
Antigens, CD4/*immunology ; Cell Line ; Cells, Cultured ; Cytokines/immunology ; Cytotoxicity, Immunologic ; Dendritic Cells/cytology/*immunology ; Humans ; Interferon-gamma/biosynthesis ; Receptors, CCR4/*immunology ; Receptors, CXCR3/*immunology ; T-Lymphocytes, Cytotoxic/*cytology/immunology ; Th1 Cells/*immunology

OBJECTIVETo construct eukaryotic expression vectors for different domains (V and VC1) of the extracellular region of the receptor of advanced glycation end products (RAGE) and investigate the roles of these domains in prostate cancer.

METHODSThe coding sequence of V and VC1 domains was amplified from the plasmid pcDNA3-HA-RAGE by PCR and cloned into the pcDNA3-HA vector following routine procedures. After identification by PCR and sequencing, the vectors including V and VC1 domains were transfected into PC-3 cells. Western blotting and immunofluorescence were used to detect the expression and distribution of the expressed products in transfected PC-3 cells.

RESULTSThe expression vectors containing V and VC1 domains of RAGE were successfully constructed as confirmed by PCR and DNA sequence analysis. The V and VC1 domains of RAGE were highly expressed and showed a cytoplasmic distribution in transfected PC-3 cells.

CONCLUSIONThe constructed eukaryotic expression vectors for V and VC1 domains of RAGE can be efficiently expressed in prostate cancer cells.

Cell Line, Tumor ; Cloning, Molecular ; Genetic Vectors ; Humans ; Male ; Plasmids ; Prostatic Neoplasms ; genetics ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo investigate the effect of Xianzhen tablet (XZT, a Chinese patent compound recipe) on advanced glycosylation end products (AGEs) and mRNA expression of AGE-specific cellular receptor (RAGE) in renal cortex of diabetic rats in order to explore the mechanism of XZT in protecting kidney.

METHODSThe diabetic rat model with persistent hyperglycemic renal damage was reproduced by streptozotocin. Fluorescent assay and RT-PCR were used to determine the content of AGEs and expression of RAGE mRNA in renal cortex in model rats, which were treated with XZT and controlled by aminoguanidine (AG) administration.

RESULTSThe relative content of AGEs and RAGE mRNA expression in renal cortex of model rats 12 weeks after modeling were significantly higher than those in the normal group (P < 0.05), while those in model rats treated by XZT or AG were markedly lower than those in non-treated model rats (P < 0.05), the effect of the both groups showed insignificant difference (P > 0.05).

CONCLUSIONXZT could reduce the accumulation of AGEs in renal cortex of diabetic rats, down-regulate the over-expression of RAGE mRNA, with the effects similar to that of AG, the inhibition of XZT on protein non-enzymatic glycosylation might be one of the mechanisms of its effect in protecting kidney.

Animals ; Diabetes Mellitus, Experimental ; genetics ; metabolism ; pathology ; Diabetic Nephropathies ; genetics ; metabolism ; pathology ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Glycation End Products, Advanced ; biosynthesis ; genetics ; Kidney Cortex ; metabolism ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; biosynthesis ; genetics ; Tablets

The study was purposed to explore the effects of NKG2D receptor and its ligands RAE-1 and H60 on graft-versus-tumor (GVT) response induced by MHC haploidentical bone marrow/spleen cell transplantation. Female (BALB/c x C57BL/6) F1 mice (CB6F1, H-2K(b/d)) inoculated with H22 cells to develop a solid tumor model were the recipients, and bone marrow mixed with spleen cells of the healthy male C57BL/6 (H-2K(b)) mice were the donor cells. GVT response was observed after transplantation that from donor cells T and NK cells were purged with anti-CD3 and anti-NK monoclonal antibody, and the NKG2D receptor was blocked with anti-NKG2D monoclonal antibody, the expression levels of RAE-1 and H60 mRNA in tumor tissue were measured by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) at different time points after transplantation. The results showed that the GVT response of transplantation was reduced after in vitro depletion of T and NK cells or blocking NKG2D receptor in donor cells of the graft, the expression levels of RAE-1 and H60 mRNA in tumor tissue increased after transplantation of haploidential bone marrow mixed with spleen cells. It is concluded that NKG2D and its ligands RAE-1 and H60 may play important roles in GVT response.
Animals ; Female ; Graft vs Leukemia Effect ; drug effects ; immunology ; Hematopoietic Stem Cell Transplantation ; Killer Cells, Natural ; drug effects ; immunology ; Leukemia, Experimental ; immunology ; therapy ; Ligands ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Minor Histocompatibility Antigens ; biosynthesis ; genetics ; NK Cell Lectin-Like Receptor Subfamily K ; Nuclear Matrix-Associated Proteins ; biosynthesis ; genetics ; Nucleocytoplasmic Transport Proteins ; biosynthesis ; genetics ; Receptors, Immunologic ; blood ; genetics ; Receptors, Natural Killer Cell

OBJECTIVETo construct a mammalian expression vector for HA-tagged receptor of advanced glycation end products (RAGE).

METHODSHuman RAGE cDNA codon region was amplified by PCR from human cDNA library and cloned into the pcDNA3 vector following routine procedures. After identification by enzyme digestion, PCR and sequencing, the correct recombinant vector RAGE/pcDNA3 was inserted with HA sequence behind the signal peptide sequence of RAGE. After identification by sequencing, HA-RAGE/pcDNA3 was transfected into HEK293 cells, and its expression was detected by Western blotting.

RESULTSIdentification by enzyme digestion, PCR and sequencing, confirmed the validity of the recombinant vector RAGE/pcDNA3, and HA-RAGE/pcDNA3 was highly expressed in HEK 293 cells.

CONCLUSIONHA-tagged RAGE is successfully constructed and expressed in mammalian cells, which may facilitate functional study of RAGE in cell signal transduction.

Animals ; Base Sequence ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Expression ; Genetic Vectors ; genetics ; Hemagglutinins ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Sequence Analysis, DNA