OBJECTIVETo measure the subsets of dendritic cells 1 (DC1) in the bone marrow of severe aplastic anemia (SAA) patients and evaluate the relationships between the CD11c+CD83+ cells and Th1 cells, CD3+CD8+ cells or hematopoietic function and explore the role of DC1 in the pathogenesis of SAA.

METHODSBy FACS, the quantities and ratios of CD11c+CD1a+ cells, CD11c+CD83+ cells, Th1 cells, and CD3+CD8+ cells in the bone marrow of SAA patients and normal controls were detected respectively. The relationships between CD3+CD8+ cells and reticulocyte absolute value (Ret) or neutrophil absolute value (ANC), between Th1 cells and CD3+CD8+ cells, Ret or ANC, between CD11c+CD83+ cells, and Th1 cells, CD3+CD8+ cells, Ret or ANC were evaluated.

RESULTSIn normal controls' bone marrow, the percentages of Th1 cells, CD11c+CD1a+ cells, CD11c+CD83+ cells and the ratio of CD11c+CD83+/CD11c+CD1a+ were (0.42 +/- 0.30)%, (0.38 +/- 0.29)%, (0.37 +/- 0.32)% and 1.07 +/- 0.10, respectively. In untreated SAA patients, they were (4.87 +/- 0.54)%, (1.73 +/- 0.24)%, (3.38 +/- 0.56)% and 2.21 +/- 0.32 respectively, which were higher than that in normal controls (P < 0.01). In recovering SAA patients, the percentages of Th1 cells, CD11c+CD1a+ cells and CD11c+CD83+ cells decreased significantly to (0.53 +/- 0.22)%, (0.61 +/- 0.23)%, (0.65 +/- 0.22)%, respectively (P < 0.01). The ratio of CD11c+CD83+/CD11c+ CD1a+ in recovering SAA patients decreased to 1.37 +/- 0.25, which was similar to that in normal controls (P > 0.05). The percentage of CD3+CD8+ cells in untreated SAA patients was (32.32 +/- 10.22)%, and in recovering SAA patients decreased to (13.67 +/- 5.24)% (P < 0.01). The percentage of CD3+CD8+ cells in SAA patients was negatively correlated with their Ret and ANC (P < 0.05), while their Th1 cell percentages were positively correlated with their CD3+CD8+ cells (P < 0.01), and negatively correlated with their Ret and ANC (P < 0.01). SAA patient's CD11c+CD83+ cell percentages were positively correlated with their Th1 cell and CD3+CD8 cells (P < 0.01, P < 0.05), but negatively with their Ret and ANC (P < 0.01).

CONCLUSIONBoth immature DC1 and activated DC1 increased in the bone marrow of SAA patients, and the balance of DC1 subsets shifted from stable form to active one, which might promote Th0 cells to polarize to Th1 cells, and cause the over-function of T lymphocytes and hematopoiesis failure in SAA.

Adolescent ; Adult ; Anemia, Aplastic ; immunology ; Antigens, CD ; immunology ; Antigens, CD1 ; immunology ; Bone Marrow ; immunology ; CD11c Antigen ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Child ; Dendritic Cells ; immunology ; Female ; Humans ; Immunoglobulins ; immunology ; Male ; Membrane Glycoproteins ; immunology ; Th1 Cells ; immunology ; Young Adult

Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1alpha, IL-1beta, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8alpha+/CD11c+ splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis.
Animals ; Antigens, CD11c/analysis ; Antigens, CD8/analysis ; Cytokines/*blood/*secretion ; Dendritic Cells/chemistry/*immunology ; Disease Models, Animal ; Flow Cytometry ; Mice ; Mice, Inbred BALB C ; Rodent Diseases/immunology ; Spleen/*immunology ; Time Factors ; Toxoplasmosis, Animal/*immunology

OBJECTIVETo investigate the effects of mobile phone 1800 MHz electromagnetic fields (EMF) on the surface markers and the functions of human dendritic cells (DC).

METHODSHuman DCs were exposed to intermittent 5 min on/10 min off EMF with specific absorption rates (SAR) 4 W/kg for 0 h, 1 h, 12 h or 24 h, respectively. FACS analysis was used to detect the positive percentage of DC surface markers including HLA-DR and co-stimulatory molecules such as CD80, CD86, CD40 and CD11c. CCK-8 kit was adopted to examine the function of allo-mixed lymphocyte reaction (allo-MLR) of DC, and enzyme linked immunosorbent assay (ELISA) to identify the levels of IL-12p70 and TNF-alpha secreted by DC.

RESULTCompared with the sham radiation group, after exposure to the electromagnetic fields for 1 h, 12 h, or 24 h, HLA-DR, CD80,CD86 and CD40 were all declined except CD11c. The ability of DC allo-MLR in each exposure group was decreased significantly (P<0.05), especially in the 24 h exposure group. However, the secreted levels of IL-12p70 and TNF-alpha of DC in each exposure group remained no changed.

CONCLUSIONThe study showed that EMF exposure could down-regulate the surface molecules and stimulation ability of human DC.

B7-1 Antigen ; B7-2 Antigen ; immunology ; Biomarkers ; analysis ; CD11c Antigen ; immunology ; Cell Phone ; Cells, Cultured ; Dendrites ; pathology ; Dendritic Cells ; metabolism ; physiology ; radiation effects ; Electromagnetic Fields ; HLA-DR Antigens ; analysis ; Humans ; Interleukin-12 ; immunology

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

OBJECTIVETo study the cytotoxic T lymphocyte (CTL) response induced by dendritic cells (DC) transduced with recombinant adenovirus vector bearing hepatitis B virus surface antigen (HBsAg) gene in hepatocellular carcinoma HepG2. 2. 15 cells in vitro.

METHODSFull length HBsAg cDNAs were subcloned into pIND vector, followed by being cloned into pShuttle vector. The HBsAg gene fragments resulted from the pShuttle-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells, to construct AdVHBsAg hepatocarcinoma tumor vaccine. The effectiveness of transfection was detected by Western blot. Surface molecules of AdVHBsAg-DC were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DC was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DC in vitro was detected by LDH assay.

RESULTSHBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. Cell pathological changes appear after 10 days HEK293 cells transfected AdVHBsAg. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DC, indicating that the transfection was effective. AdVHBsAg-DC was able to upregulate CD1a, CD11c, CD80, CD86 and HLA-DR. Autologus T cell proliferation induced by AdVHBsAg-DCs was significantly higher than that in DC control group and LacZ-DC group (P < 0.05). AdVHBsAg-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing HBsAg.

CONCLUSIONDC transduced with recombinant adenovirus HBsAg can express HBV-related hepatocellular carcinoma antigen (HBsAg), and AdVHBsAg-DC can induce potent immune response against HBsAg-positive hepatocellular carcinoma cells in vitro.

Adenoviridae ; genetics ; Antigens, CD1 ; metabolism ; CD11c Antigen ; metabolism ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; immunology ; pathology ; virology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; metabolism ; Genetic Vectors ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Humans ; Liver Neoplasms ; immunology ; pathology ; virology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

Secondary lymphoid tissue chemokine (SLC), which is expressed in T cell zones of secondary lymphoid organs, including the spleen and lymph nodes, strongly recruits both T lymphocytes and mature dendritic cells. As appropriate interaction of tumor-specific T cells and mature dendritic cells, equipped with tumor antigens, is a prerequisite for effective T cell immunity against established tumors, we mobilized lymphocytes and dendritic cells to tumor sites by intratumoral injection of secondary lymphoid tissue chemokine-Fc (SLC-Fc) fusion protein using the B16F10 murine melanoma model. Activation of dendritic cells, another prerequisite for the effective activation of naive tumor-specific T cells, was achieved by the addition of immunostimulatory cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG-ODN) into the tumor site. Intratumoral administration of SLC-Fc or CpG-ODN revealed antitumor effects against B16F10 murine melanoma grown in the subcutaneous space. Co-treatment of SLC-Fc and CpG-ODN displayed synergistic effects in reducing the tumor size. The synergistic antitumor effect in co-treatment group was correlated with the synergistic/additive increase in the infiltration of CD4+ T cells and CD11c+ dendritic cells in the tumor mass compared to the single treatment groups. These results suggest that the combined use of chemokines and adjuvant molecules may be a possible strategy in clinical tumor immunotherapy.
Animals ; Antigens, CD11c/immunology ; CD4-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chemokine CCL21/*administration & dosage/pharmacology ; Chemotaxis, Leukocyte ; Dendritic Cells/immunology/metabolism ; Immunotherapy ; Injections, Intralesional ; Melanoma, Experimental/*immunology/*therapy ; Mice ; Mice, Inbred C57BL ; Oligodeoxyribonucleotides/*administration & dosage/pharmacology ; T-Lymphocytes/immunology/metabolism

OBJECTIVETo investigate the influence of glucocorticoid on phenotype of thymic dendritic cells in mice and to investigate the protective effect of Yougui Pill (YGP) on it.

METHODSBALB/c mice allocated in the group A and B were treated respectively with 10 mg/kg hydrocortisone, alone and combined with 20.81 g/kg YGP. The control mice were treated with normal saline. The changes before and after treatment of I-A(d) and H-2K(d) antigen presentation molecules expression in CD11c(+) and CD45(+) thymic dendritic cells of mice were analyzed by flow cytometry assay, and the expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) mRNA in thymocytes were determined by RT-PCR as well.

RESULTSThe percentage of I-A(d+) and H-2K(d+) in CD11c(+) in Group A after treatment was 46.77 +/- 4.32% and 64.34 +/- 7.69% respectively, as compared with those in the control group (65.81 +/- 7.69% and 31.88 +/- 5.01%), the percentage of I-A(d+) was lower and that of H-2K(d+) was higher significantly (all P <0.01). Meantime, the expression of ICAM-1 and LFA-1 in thymocyte in Group A (30.11 +/- 2.51% and 30.40 +/- 3.77%) was significantly lower than that in the control group (46.35 +/- 3.34% and 47.28 +/- 2.91%) respectively (P <0.01). Changes in Group B showed that treated by hydrocortisone in combination with YGP, the above-mentioned hydrocortisone-induced changes could be obviously reversed, the outcome of CD11c(+) I-A(d+) was 54.19 +/- 5.08%, ICAM-1 33.97 +/- 2.04% and LFA-1 34.80 +/- 2.92%, the difference between the two treated groups in these indexes all showed statistical significance (P <0.05).

CONCLUSIONGlucocorticoidcan inhibit the expression of major histocompatibility complex class II antigen molecule, but promote the expression of major histocompatibility complex class I in CD11c(+) and CD45(+) dendritic cells, down-regulate ICAM-1 and LFA-1 transcription, while the tonifying yang recipe, YGP, has a dominant protective effect against the above actions of glucocorticoid.

Animals ; CD11c Antigen ; metabolism ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; H-2 Antigens ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Hydrocortisone ; toxicity ; Intercellular Adhesion Molecule-1 ; metabolism ; Leukocyte Common Antigens ; metabolism ; Lymphocyte Function-Associated Antigen-1 ; metabolism ; Mice ; Mice, Inbred BALB C ; Phenotype ; Protective Agents ; pharmacology ; Thymus Gland ; cytology ; drug effects ; immunology

OBJECTIVETo explore the role of lung interstitial dendritic cells in immunodissonance and organ injury in multiple organ dysfunction syndrome (MODS).

METHODSAnimal model of MODS was established by injecting zymosan into the peritoneal cavity of C57BL/6 mice. The mice were randomly divided into groups of normal, 3 - 6 hours, 12 - 48 hours, 5 - 7 days, 10 - 12 days post injection. Pathological changes of lung and interstitial dendritic cells were studied by light and transmission electron microscope. Immunohistochemistry, RT-PCR and flow cytometry analyses were used to document status of biomarkers, including specific surface markers (CD205 and CD11c), costimulatory molecules (CD80 and CD86), SLC and its receptor CCR7 in lung, CD4+ and CD8+ T lymphocyte subtypes in peripheral blood.

RESULTSAt early stage of injury, interstitial dendritic cells showed an increase in proliferation with expression of low level of CD80 and CD86. In contrast, the expression of SLC and its receptor CCR7 in lung were increased. The ratio of CD4+/CD8+ declined in peripheral blood. At the stage of SIRS, interstitial dendritic cells continued to proliferate with high expressions of CD80 and CD86. SLC and CCR7 in lung also increased. The ratio of CD4+/CD8+ declined markedly in peripheral blood. At the MODS stage, interstitial dendritic cells further proliferated, but the expression of CD80 and CD86 declined to a very low level. Although the level of SLC increased consistently, the level of CCR7 continued to decrease, along with a markedly decreased CD4+/CD8+ ratio in peripheral blood.

CONCLUSIONSAlterations of lung interstitial dendritic cells are likely to influence the course of immunological dysfunction of MODS. The level of CCR7 may serve as an indicator of the migration activity of interstitial dendritic cells and systemic immune response.

Animals ; Antigens, CD ; metabolism ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD11c Antigen ; metabolism ; CD4-CD8 Ratio ; Cell Proliferation ; Chemokine CCL21 ; metabolism ; Dendritic Cells ; immunology ; metabolism ; ultrastructure ; Disease Models, Animal ; Lectins, C-Type ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Minor Histocompatibility Antigens ; Multiple Organ Failure ; chemically induced ; immunology ; metabolism ; pathology ; Random Allocation ; Receptors, CCR7 ; metabolism ; Receptors, Cell Surface ; metabolism ; Zymosan