Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.
Antibodies, Bacterial/immunology ; Antigens, CD11c/metabolism ; Antigens, CD14/metabolism ; Antigens, CD18/metabolism ; Apoptosis/*immunology ; Biological Assay ; Cell Differentiation ; Cell Line, Tumor ; Cholecalciferol/pharmacology ; Dimethylformamide/pharmacology ; Flow Cytometry ; HL-60 Cells ; Humans ; Phagocytosis/*immunology ; Pneumococcal Vaccines/*immunology ; Receptors, IgG/metabolism ; Receptors, Immunologic/*biosynthesis ; Respiratory Burst/immunology ; Streptococcus pneumoniae/*immunology ; Tretinoin/pharmacology

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.
Antibodies, Bacterial/immunology ; Antigens, CD11c/metabolism ; Antigens, CD14/metabolism ; Antigens, CD18/metabolism ; Apoptosis/*immunology ; Biological Assay ; Cell Differentiation ; Cell Line, Tumor ; Cholecalciferol/pharmacology ; Dimethylformamide/pharmacology ; Flow Cytometry ; HL-60 Cells ; Humans ; Phagocytosis/*immunology ; Pneumococcal Vaccines/*immunology ; Receptors, IgG/metabolism ; Receptors, Immunologic/*biosynthesis ; Respiratory Burst/immunology ; Streptococcus pneumoniae/*immunology ; Tretinoin/pharmacology

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

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

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 explore the effect of HMGB1 on the VEGF-C expression and proliferation of esophageal squamous cancer cells as well as its possible mechanism.

METHODSA cassette encoding siRNA targeting HMGB1 mediated by rAAV was constructed, the rAAV-siHMGB1-hrGFP, and a vector encoding siRNA mismatching HMGB1 was constructed, the rAAV-miHMGB1-hrGFP. This experiment in vitro included three groups, namely, the blank control group (group A) of KYSE150 cells transfected by rAAV-hrGFP, negative mismatch control group (group B) of KYSE150 cells transfected with rAAV-miHMGB1-hrGFP, and RNA interference group (group C) of KYSE150 cells transfected with rAAV-siHMGB1-hrGFP. We examined the expression of HMGB1 mRNA and protein in the three group cells by real-time PCR and Western blot after 24 h and 48 h, respectively. Then, VEGF-C expression and cell proliferation in the three group cells with or without sRAGE, as an inhibitor of RAGE signal pathway, were assayed by ELISA and MTT after 24 h.

RESULTSThe expression of HMGB1 mRNA and protein in KYSE150 cells in vitro in the group C transfected with rAAV-siHMGB1-hrGFP at the final concentration of 2×10(6) v.g/cell was significantly lower than that of the group A or B after 24 h and 48 h (P < 0.01). The VEGF-C expression of KYSE150 cells was (502.43 ± 13.10) pg/ml in the group C, significantly reduced in comparison with that of the group A (686.40 ± 10.94) pg/ml or group B (682.31 ± 9.61) pg/ml after 24 h (P < 0.05). At the same time, the proliferation of KYSE150 cells in the group C was significantly inhibited compared with that of groups A and B after 24 h (P < 0.01). Moreover, sRAGE at the final concentration of 0.2 µg/ml inhibited the VEGF-C expression and proliferation of KYSE150 cells compared with the corresponding group without sRAGE after 24 h (P < 0.01 or P < 0.05). However, there was no significant difference of the VEGF-C expression and proliferation of KYSE150 cells with sRAGE in the group C compared with that of cells with sRAGE of the group A or group B after 24 h (P > 0.05).

CONCLUSIONSIn esophageal squamous cell carcinoma, HMGB1 can promote the VEGF-C expression and proliferation of the cancer cells through RAGE signal pathway, and HMGB1-RAGE may become a potential target for cell proliferation and lymph node metastasis of this cancer.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Dependovirus ; genetics ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HMGB1 Protein ; biosynthesis ; genetics ; Humans ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor C ; metabolism

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

We studied the immunogenicity of pseudorabies virus gC DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gC gene was linked to four copies of C3d receptor binding domain (M284), and then cloned into the vector pcDNA3.1 to construct the recombinant plasmid sgC-M284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgC-M284 was 17-fold higher than that for sgC alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD50) challenge. In addition, the IL-4 levels for sgC-M284 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production.
Adjuvants, Immunologic ; physiology ; Animals ; Antibody Formation ; immunology ; Binding Sites ; Cloning, Molecular ; Complement C3d ; genetics ; immunology ; Herpesvirus 1, Suid ; genetics ; immunology ; Interleukin-4 ; immunology ; Mice ; Mice, Inbred BALB C ; Pseudorabies Vaccines ; immunology ; Receptors, Complement 3d ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; pharmacology ; Viral Fusion Proteins ; immunology

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 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