Antigens, CD1 ; physiology ; Cicatrix, Hypertrophic ; etiology ; immunology ; HLA-DR Antigens ; physiology ; Humans ; Keloid ; etiology ; immunology ; Triamcinolone Acetonide ; pharmacology

The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective antigen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34+ cells collected from healthy donors. CD34+ cells purified from leukapheresis product were seeded at 1 x 10(4) cells/mL in complete medium supplemented with GM-CSF, TNF alpha, IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CSF + TNF alpha, the total cell number increased by 23.4 +/- 5.4-fold compared to the starting number of CD34+ cells. When the c-kit and FL were added to GM-CSF and TNF alpha, the cell number increased by 109.8 +/- 11.2-fold without affecting the immunophenotype of recovered cells. Flow cytometric analysis indicated that cells with the markers of mature dendritic cells, i.e., CD1a +CD14 -HLA-DR+, and CD80+CD86+HLA-DR+, constituted 49.0% +/- 7.5%, and 38.9% +/- 6.5%, respectively. This pattern of expression of surface antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a+HLA-DR+ cells recovered from in vitro cultures elicit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespective of cytokine combinations. These findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.
Antigens, CD34/analysis* ; Dendritic Cells/physiology* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology ; HLA-DR Antigens/analysis ; Hematopoietic Stem Cells/physiology* ; Human ; Interleukin-4/pharmacology ; Tumor Necrosis Factor/pharmacology

OBJECTIVE: To investigate the effects of dasatinib on the maturation of monocyte-derived dendritic cells (moDCs) derived from healthy donors (HDs) and chronic myelogenous leukemia (CML) patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from HDs (n=10) and CML patients (n=10) who had got the remission of MR4.5 with imatinib treatment. The generation of moDCs from PBMCs was completed after 7 days of incubation in DC I culture medium, and another 3 days of incubation in DC II culture medium with or without 25 nmol/L dasatinib. On the 10th day, cells were harvested and expression of molecules of maturation related marker were assessed by flow cytometry. The CD80⁺CD86⁺ cell population in total cells was gated as DCs in the fluorescence-activated cell storting (FACS) analyzing system, then the expression of CD83, CD40 or HLA-DR in this population was analyzed respectively. RESULTS: The proportion of CD80⁺CD86⁺ cells in total cells didn't show a statistical difference between HD group and patient group (89.46%±9.70% vs 87.39%±9.34%, P=0.690). Dasatinib significantly enhanced the expression of the surface marker CD40 (P=0.008) and HLA-DR (P=0.028) on moDCs derived from HDs compared with the control group, while the expression of CD83 on moDCs didn't show a significant difference between dasatinib group and the control group (P=0.428). Meanwhile, dasatinib significantly enhanced the expression of the surface marker CD40 (P=0.023), CD83 (P=0.038) and HLA-DR (P=0.001) on moDCs derived from patients compared with the control group. CONCLUSION For CML patients, the same high proportion of moDCs as HDs can be induced in vitro, which provides a basis for the application of DC-based immunotherapy strategy. Dasatinib at the concentration of 25 nmol/L can efficiently promote the maturation of moDCs derived from HDs and CML patients in vitro. Dasatinib shows potential as a DC adjuvant to be applied in DC-based immunotherapy strategies, such as DC vaccine and DC cell-therapy.
Cell Differentiation ; Cells, Cultured ; Dasatinib/pharmacology* ; Dendritic Cells ; HLA-DR Antigens/pharmacology* ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism* ; Leukocytes, Mononuclear ; Monocytes

Gamma interferon (gamma-IFN), a lymphokine produced by activated T lymphocytes, has a variety of effects on target cell. It induces class II antigens of the major histocompatibility complex not only in immunocompetent cells but also in non-immunocompetent cells. gamma-IFN also can exert, in addition to anti-viral activity, a series of anticellular effects on a variety of cell types. The effects of gamma-IFN on the proliferation of cultured epidermal cell (EC) and induction of HLA-DR antigen expression by EC (HLA-DR+KC) were studied. Furthermore, the immunologic role of HLA-DR+KC in the mixed epidermal cell-lymphocyte reaction (MECLR) was studied. The antiproliferative effect of gamma-IFN on the cultured EC was seen 3 days after treatment of gamma-IFN and the effect was dose-dependent. Number of HLA-DR+KC was increased dose-dependently with treatment of gamma-IFN. In MECLR, HLA-DR+KC had been found to exert stimulatory role on allogenic lymphocytes. However, there was no significant role of HLA-DR+KC on autologous lymphocytes.
Adolescent ; Adult ; Cell Division/*drug effects ; Female ; HLA-DR Antigens/*immunology ; Humans ; Interferon-gamma/*pharmacology ; Lymphocytes/cytology/drug effects/*immunology ; Male ; Skin/*cytology/drug effects

OBJECTIVETo investigate the effect of synthetic non-methylated CpG-ODN combined with recombined HBsAg on the phenotype, function and the expression level of nucleic transcription factor NF-kappa B (NF-kB) and AP-1 of monocyte-derived dendritic cells (DC) in patients with chronic hepatitis B (CHB).

METHODSPurified monocytes were isolated from the peripheral blood of CHB patients and healthy volunteers and cultured with human granulocyte-monocyte colony stimulating factor added together with interleukin-4. On the fifth day of culture, CpG-ODN, HBsAg and other reagents, such as TNF alpha and PBS, were added to the medium, and 18 hours later cells were collected for the detection of surface molecules (HLA-DR/CD86/CD1a). IL-12p70 levels in the supernatant and stimulating capacity to allogenic T lymphocytes were detected. The nucleic proteins of NF-kB and AP-1 in DC were extracted and purified for the gel shift assay.

RESULTSCompared with those of the PBS group, the expression rates of HLA-DR of DC treated with CpG-ODN and/or HBsAg were obviously increased. Both the IL-12p70 level and the stimulating capacity of DC to allogenic T lymphocytes in mixed lymphocyte reaction increased significantly in the CpG-ODN group and in the CpG-ODN/HBsAg combination group (P less than 0.01 and 0.05, respectively). The expression rates of CD1a were raised only in the CpG-ODN/HBsAg combination group. Three kinds of immunological adjuvants, TNF alpha, CpG-ODN and CpG-ODN/HBsAg enhanced the expression of nucleic NF-kB and inhibited the expression of AP-1 in DC.

CONCLUSIONCpG-ODN, like TNF alpha, has remarkable stimulatory effect on the impaired phenotype and function of monocyte-derived DC in patients with CHB; CpG-ODN and HBsAg have a synergetic effect in increasing the antigen presenting function. The regulating ability of CpG-ODN and TNF alpha on the expression levels of NF-kB and AP-1 might be one of the mechanisms of their immunostimulatory effects on DC of the CHB patients.

Adjuvants, Immunologic ; pharmacology ; Adult ; Cells, Cultured ; Dendritic Cells ; metabolism ; HLA-DR Antigens ; metabolism ; Hepatitis B Surface Antigens ; pharmacology ; Hepatitis B, Chronic ; metabolism ; Humans ; Male ; NF-kappa B ; metabolism ; Oligodeoxyribonucleotides ; pharmacology ; Recombinant Fusion Proteins ; Transcription Factor AP-1 ; metabolism

This study investigated the feasibility of using an hammerhead ribozyme against C II TA, a major regulator of MHC II antigens, to repress the expression of MHC II molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463-465 GUC triplet of C II TA and its target gene were transcribed, then mixed up and incubated in vitro. The cleavage products were analyzed by PAGE and silver-staining. Rz464 was then inserted into the pIRES2-EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class II MHC induction by recombinant human interferon-gamma (IFN-gamma). mRNA of C II TA was measured by RT-PCR. Our results showed that Rz464 could exclusively cleave C II TA RNA. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on pRz464+ Hela was induced, and the mRNA content of C II TA decreased too. It is concluded that Rz464 could inhibit C II TA and thus the family of genes was regulated by C II TA:MHC II molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto-immune diseases.
Autoimmune Diseases ; therapy ; Base Sequence ; Genetic Therapy ; HLA-DP Antigens ; metabolism ; HLA-DQ Antigens ; metabolism ; HLA-DR Antigens ; metabolism ; HeLa Cells ; Humans ; Interferon-gamma ; pharmacology ; Molecular Sequence Data ; Nuclear Proteins ; immunology ; RNA, Catalytic ; genetics ; metabolism ; pharmacology ; physiology ; Recombinant Proteins ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; antagonists & inhibitors ; genetics ; immunology ; Transfection

UNLABELLEDDendritic cells (DC) are very potent antigen-presenting cells (APC) with a unique ability to activate naive T cells to induce the differentiation of TH1/TH2. Monocytes can develop into DC in the presence of different cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. DCs are thought to play a key role in the initiation and maintenance of T cell immunity to inhaled antigens. While the density of DC within the bronchial mucosa is increased in asthma, there is little information currently available concerning the effects of DC in asthmatic children.

OBJECTIVETo investigate the role of dendritic cells in the pathogenesis of acute attack of asthma in children.

METHODSThomas' method was adopted. The adherent precursors of DC were isolated from peripheral blood of asthmatic children in acute attack stage and healthy controls. The adherent cells were induced with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha) to DC in vitro. The expression of the surface molecules CD80, CD86, HLA-DR etc. on the DC was examined by fluorescent activated cell sorter (FACS). And the ability to secret IL-10, IL-12 and their potentials to stimulate the proliferative reaction of DC inductive self T- lymphocyte were observed.

RESULTSThe results showed that in asthmatic children's acute attack stage, self T- lymphocyte proliferative reaction induced by DC was remarkably increased compared with normal control subjects (P < 0.01). At the same time, the asthmatic children in acute attack stage had remarkably decreased the ability to secret IL-10 compared with normal control subjects (P < 0.01), while the ability to secret IL-12 remarkably decreased compared with normal control subjects (P < 0.01); meanwhile, the HLA-DR and co-stimulating factor CD86(B(7-2)) expressed by DCs remarkably increased in the asthmatic children in acute attack stage compared with normal control subjects (P < 0.01).

CONCLUSIONDC possibly plays a vital role in the immunological mechanism of asthma by means of inducing the differentiation of TH1/TH2, that is DC may be the key factor in initiating the airway allergic reaction and the possible mechanism may involve interleukins (especially IL-10 and IL-12, etc.) secreted by DCs.

Adolescent ; Antigens, CD ; metabolism ; Asthma ; metabolism ; physiopathology ; Child ; Child, Preschool ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HLA-DR Antigens ; metabolism ; Humans ; Interleukin-10 ; secretion ; Interleukin-12 ; secretion ; Interleukin-4 ; pharmacology ; Male

To investigate whether the dendritic cells (DC) could grow up in cultural system with umbilical cord serum (UCS), the UCS was used in the culture instead of fetal calf serum. The phenotype of dendritic cells was detected by flow cytometry and the antigen presenting ability of DC in allo-MLR was measured by MTT assay. The results showed that DC grown in UCS (UCS-DC) had higher expression rate of CD86, CD83 and HLA-DR than that in grown in FCS (FCS-DC). (P < 0.05), and their expression of CD1a was lower than that of FCS-DC. The ability to induce T cell proliferation had no difference between UCS-DC and FCS-DC. It is suggested that dendritic cells with more mature phenotype had been produced in the medium containing UCS than those in the medium containing FCS, and UCS-DC possessed potent ability to stimulate proliferation of allogeneic T cells.
Animals ; Antigens, CD ; immunology ; B7-2 Antigen ; Cattle ; Cell Culture Techniques ; methods ; Cell Division ; drug effects ; Culture Media ; pharmacology ; Dendritic Cells ; cytology ; drug effects ; immunology ; Fetal Blood ; physiology ; HLA-DR Antigens ; immunology ; Humans ; Immunoglobulins ; immunology ; Membrane Glycoproteins ; immunology

Antigen presenting is the initial step of the immune responses. In order to verify that human bronchial epithelial cells (HBECs) can express antigen presentation molecules, which can be modulated by intrapulmonary regulatory peptides, the present study was designed to examine the expressions of human leukocyte antigen DR (HLA-DR), CD80 and CD86 in resting or ozone-stressed HBECs by using immunocytochemistry and flow cytometry analysis. The results showed that HBECs expressed HLA-DR, CD80 and the expressions of HLA-DR and CD80 molecules were down-regulated under ozone stress. While VIP, P3513 and CGRP upregulated the expression of HLA-DR in resting or ozone-stressed HBECs, they had different effects on CD80 expression. VIP did not influence the expression of CD80 under resting state, but increased the expression of CD80 under ozone stress. CGRP decreased CD80 expression in resting HBECs, but increased CD80 expression in ozone-stressed HBECs. P3513 increased CD80 expression in resting HBECs, but decreased CD80 expression in ozone-stressed HBECs. The expression of CD86 was absent in resting or ozone-stressed HBECs. The results obtained demonstrate that HBECs have the capability to act as antigen presenting cells and the expression of HLA-DR and costimulatory molecules can be modulated by intrapulmonary regulatory peptides.
Antigen-Presenting Cells ; metabolism ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Bronchi ; cytology ; Calcitonin Gene-Related Peptide ; pharmacology ; Epithelial Cells ; metabolism ; HLA-DR Antigens ; metabolism ; Humans ; Ozone ; adverse effects ; Vasoactive Intestinal Peptide ; pharmacology

The present study was purposed to investigate the relation and difference of expression phase between class II transactivator (CIITA) and HLA-DR antigens after IFN-gamma induction, and the inhibition of CIITA and HLA-DR by STAT1-alpha antisense oligonucleotides (STAT1-alpha AS); and to explore the potential effect and significance of CIITA and STAT1-alpha AS in transplantation immunity. T lymphocytes from peripheral blood of healthy subjects were incubated with IFN-gamma at different doses. RT-PCR was used to detect CIITA mRNA and Western blot was used to analyze HLA-DR antigen. Then the optimum dose of IFN-gamma was chosen for the experiment. CIITA mRNA and HLA-DR antigen were detected at various time points. Different doses of STAT1-alpha AS and sense oligonucleotides (STAT1-alpha S) were added to T lymphocytes followed by IFN-gamma. After incubation with IFN-gamma, the expression of CIITA mRNA and HLA-DR was detected once again. The results showed that CIITA mRNA was detectable at 5 hours after IFN-gamma incubation and reached the peak at 14 hours, then declined, but the CIITA mRNA was still found at 23 hours. HLA-DR antigen was detectable at 28 hours after IFN-gamma incubation and reached a peak at 52 hours, then declined. CIITA mRNA expression was positively correlated to HLA-DR expression, and was earlier than the latter. The expression of CIITA mRNA in the AS groups was significantly lower than that in the control group after 5 micromol/L, 10 micromol/L and 20 micromol/L STAT1-alpha AS treatment (P < 0.01). The expression of CIITA mRNA in the S groups was higher than that in the AS groups (P < 0.01), but there was no significant difference between the S group and the control group. The expression of HLA-DR antigen was significantly inhibited by STAT1-alpha AS, and the expression level of HLA-DR protein in the AS group was about 64.3% of that in the control group (P < 0.01), while there was no significant difference in HLA-DR expression between the S group and the control group. The changes in HLA-DR expression were similar to those in CIITA expression after STAT1-alpha AS treatment. It is concluded that CIITA expression is positively correlated with HLA-DR expression, and was detectable earlier than that of latter after IFN-gamma incubation. Stat1-alpha antisense oligonucleotides may have a sequence-specific inhibiting effect on the expression of CIITA and HLA-DR antigen after IFN-gamma incubation in vitro culture, and can prevent T lymphocyte activation. CIITA may play an important role in pathogenesis of transplantation immunity.
Cells, Cultured ; HLA-DR Antigens ; biosynthesis ; genetics ; Humans ; Interferon-gamma ; pharmacology ; Nuclear Proteins ; biosynthesis ; genetics ; Oligonucleotides, Antisense ; antagonists & inhibitors ; RNA, Messenger ; biosynthesis ; genetics ; STAT1 Transcription Factor ; antagonists & inhibitors ; T-Lymphocytes ; cytology ; Trans-Activators ; biosynthesis ; genetics ; Transplantation Immunology ; immunology