Functional disruption of human leukocyte antigen II in human embryonic stem cell

BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allo-graft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA(-/-)hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA(-/-)hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA(-/-)hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA(-/-)hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA(-/-)hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4+ T cells, which are the main effector cells of cellular immunity in allograft.

Study of Trichostation A-Induced Expression of Costimulatory Molecules CD80 and CD86 in Acute Myelocytic Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the trichostain A (TSA)-induced expression of costinmulatory molecules CD80 and CD86 in HL-60, K562 and mononuclear cells (MNC) of bone marrow in AML patients and its clinical significance.</p><p><b>METHODS</b>The TSA-induced expression of costimulatory molecules CD80, CD86 in HL-60, K562 and BMMNC, and the cell viability were detected by flow cytometry; the mRNA expression of CD80 and CD86 was detected by RT-PCR; after the TSA-induced HL-60 cells and K562 cells were irradiated with 75 Gy, the effect of these cells on proliferation of PBMNC from healthy volunteers was determined with CCK-8 method.</p><p><b>RESULTS</b>The HL-60 cells and BMMNC in AML patients expressed CD86, not expressed CD80, while the K562 cells not expressed CD86 and CD80. TSA could up-regulate the expression of CD86 in HL-60 cells and BMMNC of AML patients. The TSA-induced HL-60 cells expressing costimulatory molecule CD86 showed the proliferative effect on BMMNC from healthy volunteers.</p><p><b>CONCLUSION</b>The TSA can induce the expression of costimulatory molecule CD86 in HL-60 cells and BMMNC in AML patients, and can improve the proliferation of PBMNC in healthy volunteers.</p>

Role of glycogen synthase kinase 3β in maturation and function of murine myeloid dendritic cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of glycogen synthase kinase 3β (GSK-3β) in the maturation and function of murine bone marrow-derived dendritic cells (BMDCs).</p><p><b>METHODS</b>Mature DCs (mDCs) induced by LPS were examined for GSK-3β phosphorylation level with Western blotting before and after LPS exposure. To explore the role of GSK-3β in maturation and function of DCs, we added SB216763, a selective inhibitor of GSK-3β, in the cell culture of immature DCs (iDCs), and examined CD40 and CD86 expressions in the cells by flow cytometry and the expression of IL-6, IL-12 and IL-10 mRNA by real-time PCR; the changes of the immunogenicity of the cells was evaluated by mixed lymphocyte reaction. The expression of GSK-3β and RelB was examined by Western blotting in DC2.4 cells transfected with a lentiviral vector over-expressing murine GSK-3β gene.</p><p><b>RESULTS</b>LPS exposure significantly lowered GSK-3β activity in iDCs as demonstrated by increased Ser9 phosphorylation and reduced Tyr216 phosphorylation. GSK-3β inhibition induced DC maturation by increasing the expression of surface costimulatory molecules CD40 and CD86, lowered the expressions of IL-6 and IL-12 while enhanced the expression of IL-10 in iDCs, and impaired mixed lymphocyte reaction of the cells. In DC2.4 cells, lentivirus-mediated over-expression of GSK-3β obviously down-regulated the expression of RelB.</p><p><b>CONCLUSIONS</b>GSK-3β is a crucial enzyme involved in the differentiation and maintenance of an immature phenotype of DCs. GSK-3β is constitutively active in iDCs to inhibit their spontaneous maturation. DCs become phenotypically mature after inhibition of GSK-3β, which also executes a proinflammatory task in DC activation. The reduction of RelB protein levels as a result of GSK-3β overexpression supports GSK-3β as a new target for inducing tolerogenic DCs.</p>

Expression of costimulatory molecule CD86 in HL-60 cells induced by MG132 and its effect on allogeneic mixed lymphocyte reaction / 中国实验血液学杂志

This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1×10(5) (P < 0.01). No remarkable proliferation of PBMNC was observed in the K562 groups no matter if the HL-60 cells had been treated with MG132. It is concluded that the high concentration of MG132 can directly kill HL-60 cells, low-concentration of MG132 can induce the expression of costimulatory molecule CD86 in HL-60 cells, also can improve the proliferation of PBMNC.

Role of Toll-like receptor 2/4-nuclear factor-κB signaling pathway in invasion of Mycobacterium tuberculosis to mouse dendritic cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells (DC).</p><p><b>METHODS</b>Mycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB(NF-κB) was assessed by Western blotting.The extracellular concentration of tumor necrosis factor α(TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion.Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2.4 cells before and after invasion.</p><p><b>RESULTS</b>The invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9±5.6)%,(51.2±7.6)%,(57.2±8.9)% and(63.9±6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-α were higher in DC2.4 cells after being invaded by 6,8,and 10 h and then gradually decreased.CD80 and CD86 expression were increased on DC2.4 at 6 h after co-incubation.</p><p><b>CONCLUSION</b>Invasion of Mycobacterium tuberculosis strain H37Rv to DC might enhance its antigen-presenting function through activation of TLR2/4-NF-kB signaling pathway.</p>

The immunostimulatory effects of retinoblastoma cell supernatant on dendritic cells

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs' antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and DC-based immunotherapy may be adopted in the treatment of RB.

Tanshinone II A inhibits dendritic cell-mediated adaptive immunity: potential role in anti-atherosclerotic activity / 中国结合医学杂志

<p><b>OBJECTIVE</b>Antigen-presenting cells such as monocytes and dendritic cells (DCs) stimulate T-cell proliferation and activation during adaptive immunity. This cellular interaction plays a role in the growth of atherosclerotic plaques. Tanshinone II A (TSN) had been shown to decrease the growth of atherosclerotic lesions. We therefore investigated the ability of TSN to inhibit human monocyte-derived DCs and their T-cellstimulatory capacity.</p><p><b>METHODS</b>DCs derived from human monocytes cultured with recombinant human interleukin (IL)-4 and recombinant human granulocyte-macrophage colony-stimulating factor were co-cultured with TSN and lipopolysaccharide for 48 h. Phosphate-buffered saline was used as a negative control. Activation markers and the capacity of DCs for endocytosis were measured by flow cytometry, and proinflammatory cytokines were measured by enzyme-linked immunosorbent assays. DCs were co-cultured with lymphocytes to measure T-cell proliferation and IL-2 secretion by mixed lymphocyte reactions.</p><p><b>RESULTS</b>TSN dose-dependently attenuated DC expression of costimulatory molecules (CD86), and decreased expression of major histocompatibility complex class II (human loukocyte antigen-DR) and adhesion molecules (CD54). Moreover, TSN reduced secretion of the proinflammatory cytokines IL-12 and IL-1 by human DCs, and restored the capacity for endocytosis. Finally, TSN-preincubated DCs showed a reduced capacity to stimulate T-cell proliferation and cytokine secretion.</p><p><b>CONCLUSIONS</b>TSN inhibits DC maturation and decreases the expression of proinflammatory cytokines, while impairing their capacity to stimulate T-cell proliferation and cytokine secretion. These effects may contribute to the influence of TSN on the progression of atherosclerotic lesions.</p>

Effects of platelet microparticles on the activation of B cells

Platelets are anucleated fragments derived from megakaryocytes. It has been demonstrated that platelets play a role in hemostasis and innate immunity. In addition, platelets have a CD40 ligand which is an important molecular marker in motivating immune cells. Thus, platelets also have a role in adaptive immunity as seen by their ability to activate B cells. Since human platelet microparticles [MPs] originate from platelets, we have chosen to examine the effects of MPs on B cell activation. Platelet MPs were isolated from platelet concentrates obtained from the Tehran Blood Transfusion Center. The MPs were co-cultured with B cells isolated from human whole blood with magnetic beads using negative selection. After seven days, the expression of activation markers CD27 and CD86, as well as IgD were evaluated by flow cytometry. In a comparison between test [B cells/MPs] and control [B cells] cells we observed that the expression of activation markers CD27 and CD86 increased during the seven-day co-culture period. However, the expression of IgD antibody decreased. As with platelets, MPs can affect B cell activation during in vitro co-culture

Association of co-stimulatory human B-lymphocyte antigen B7-2 [CD86] gene polymorphism with colorectal cancer risk

This study investigated the role of CD86 +237 G/C polymorphism in intensifying the risk of CRC development. Colorectal cancer [CRC] is a multi-factorial diseases. Genetic background could affect the susceptibility of individuals to CRC development. CD86 is a co-stimulatory factor on antigen-presenting cells that plays key roles in several cancer related mechanisms such as autoimmunity, transplantation and tumor immunity. A total of 300 individuals, 150 known CRC patients and 150 healthy control individuals, were subjected for the study. CD86 rs17281995 single nucleotide polymorphism [SNP] was genotyped using Allelic Discrimination method. A statistically significant difference was found among CD86 gene polymorphism [rs17281995] and risk of CRC development. The frequency of GG, GC and CC in control subjects was determined as 38%, 57.3% and 4.7% respectively and in CRC subjects were determined as 42%, 85% and 23% respectively. The data shows a significant association between CC genotype [P=0.007] and C allele [P=0.017] of the studied polymorphism and risk of CRC. CC genotype and C allele are also more frequent in female patients when the data is stratified according to gender status. Our results suggest that CD86 gene alteration could affect the individual's risk for developing CRC among Iranian population and could be used as an important prognostic factor associated with risk of CRC

Recipient dendritic cells modified by RNA interference targeting CD80 and CD86 elicit T cell hyporesponsiveness via enhanced T cell apoptosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Despite extensive research, the mechanism of immature dendritic cells (DCs) induced immune hyporesponsiveness remains incompletely understood.</p><p><b>METHODS</b>Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin (IL)-2, IL-4, IL-10, and interferon (INF)-γ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining.</p><p><b>RESULTS</b>There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups (P < 0.05), indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups (P < 0.05). There was no significant difference in IL-4 levels between the groups (P > 0.05). We also showed that CD80/86 low DCs loaded with alloantigen (1) stimulated low T cell proliferative responses via the indirect recognition pathway and (2) enhanced apoptotic activity (P < 0.05) in co-cultured T cells.</p><p><b>CONCLUSIONS</b>Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinically applicable.</p>

Distribution and activation of dendritic cells in immune thrombocytopenia patients / 中国实验血液学杂志

Defective dendritic cell (DC) functions have been implicated in ITP. The purpose of this study was to investigate the distribution and activation of dendritic cells in immune thrombocytopenia (ITP) patients. ITP patients were divided into 3 groups: the newly diagnosed, refractory and effective treatment group. The distributions of plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) in peripheral blood, bone marrow and spleen were detected with flow cytometry. The expression level of CD80 and CD86 on surface of pDC and mDC was also detected with flow cytometry. The results indicated that the percentage of mDC was higher than that of pDC in all sites of all groups. The percentage of mDC and pDC in all site of refractory group was higher than that in newly diagnosed and effective groups, but the percentage of mDC in spleen of refractory group was obviously higher than that in other sites. The percentage of pDC was no significant different in all groups. The expression level of CD86 in all groups was higher than that of CD80, the expression level of CD80 was lower in mDC and pDC of all groups, but there was no obvious difference in all sites. The CD86 expression in all site of refractory group was higher than that in newly diagnosed and effective treatment groups, while the CD86 expression of mDC in spleen of newly diagnosed group obviously higher than that in other sites. It is concluded that the distribution abnormality of mDC and pDC exists in ITP patients, the mDC are more accumulated in spleen, and differentiation of mDC to maturity is more obvious in spleen, spleen-derived mDC significantly express CD86, spleen-derived mDC may play an important role in the pathogenesis of ITP.

Dendritic cell subsets and function in newborns from mothers of different HBV infection status / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The aim of this study was to explore the frequency of mDC and pDC and expression of surface markers of the neonates and to discuss the effect of different status of HBV infection of mother on biological characteristics of DC.</p><p><b>METHODS</b>Umbilicus cord blood in neonates of HBeAg positive HBV infected mother, HBeAg negative HBV infected mother, and normal mother were collected respectively; peripheral blood of healthy adults were selected as control group. Flow cytometry was employed to detect frequency of the mDC and its expression of CD86, frequency of pDC and its expression of CD80, CD83, CD86, and FlowJo software was used to compare these indicators among the groups.</p><p><b>RESULTS</b>Compared with control group, the frequency of mDC of cord blood (0.29 +/- 0.16 vs 0.81 +/- 0.17), CD86 positive rate of mDC (10.72 +/- 10.01 vs 32.13 +/- 7.46), the frequency of pDC (0.15 +/- 0.07 vs 0.30 +/- 0.07), and CD86/CD83 positive rate of pDC (31.61 +/- 12.81 vs 74.96 +/- 9.78; 42.66 +/- 20.83 vs 82.00 +/- 6.94) were lower (t = -7.86, P = 0.00; t = -5.36, P = 0.00; t = -5.43, P = 0.00; t = -8.49. P = 0.00; t = -4.90, P = 0.00).</p><p><b>CONCLUSIONS</b>The frequency of mDC and pDC in umbilical cord blood was lower than the peripheral blood of healthy adult, which was the possible mechanism of newborns easier to chronicity after the infection of hepatitis B virus. A significant correlation was found between different status of HBV infection and costimulatory molecule CD86 positive rate of mDC, but not for the frequency of mDC and pDC, and the expression of pDC molecules.</p>

Effects of Bushen Jiedu Recipe and Jianpi Jiedu Recipe containing plasma on dendritic cells of chronic hepatitis B virus infection patients under different immune states / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To compare the effects of Bushen Jiedu Recipe (BJR) and Jianpi Jiedu Recipe (JJR) containing plasma on dendritic cells (DCs) of chronic hepatitis B virus (HBV) infection patients under different immune states.</p><p><b>METHODS</b>Recruited were 36 chronic HBV infection outpatients from First Affiliated Hospital of Hunan University of Traditional Chinese Medicine from April 2010 to January 2011. They were assigned to the immune tolerance group (18 cases) and the immune clearance group (18 cases).Another 10 healthy subjects were recruited as the healthy control group. Their anticoagulated peripheral venous blood was respectively collected. The peripheral blood mononuclear cells (PBMCs) were isolated and further extracted for incubating DCs. The DCs were intervened by BJR and JJR containing plasma. The morphology of DCs was identified. The expressions of CD1alpha, CD80, CD86, and HLA-DR were detected. The level of interferon-alpha (IFN-alpha) in the supernatant was observed by ELISA.</p><p><b>RESULTS</b>The CD80 expression level was lower in the immune clear group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, CD86, and HLA-DR were lower in the immune tolerance group than in the healthy control group before intervention (P < 0.05).The IFN-alpha expression level was lower in the immune tolerance group and the immune clearance group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, HLA-DR, and IFN-alpha were lower in the immune tolerance group than in the immune clearance group before intervention (P < 0.05). Compared with the same group before intervention, the CD80 expression significantly increased in each treatment group (P < 0.05). After intervention the expression levels of CD80 and HLA-DR were higher in the immune tolerance group than in the immune clearance group in the same time phase, and the CD86 expression level was higher in the BJR group than in the immune clearance group in the same time phase, showing statistical difference (P < 0.05).</p><p><b>CONCLUSIONS</b>The middle dose BJR and the small dose JJR both could promote the recovery of DCs in chronic HBV infection patients. Besides, BJR showed more prominent effects on the function of DCs in chronic HBV infection patients in the immune tolerance stage.</p>

Inducing maturation of monocyte-derived dendritic cells on human epithelial cell feeder layer

Nowadays, dendritic cells [DCs] have a special place in cancer treatment strategies and they have been used for tumor immunotherapy as they can induce immune response against tumor cells. Researchers have been trying to generate efficient dendritic cells in vitro; therefore, this research was done to generate them for use in research and tumor immunotherapy. This study took place at Urmia University in 2010-2011 years. In this study plastic adherent monocytes were incubated with granulocyte-macrophage colony stimulating factor [GM-CSF] and interleukin-4 [IL-4] for five days. Finally, fully matured and stable DCs were generated by 48 hours of incubation in a monocyte conditioned medium [MCM] containing tumor necrosis factor-alpha [TNF-alpha] and epithelial cells. Phenotypic and functional analysis were carried out by using anti-CD 14, anti-CD80, anti-CD86, and anti-CD83 monoclonal antibodies, and by determining their phagocytic activity, mixed lymphocyte reaction [MLR] and cytokine production, respectively. Dendritic cells were produced with high levels of surface molecule, i.e. of CD80, CD83, CD86, HLA-DR, expression and low levels of CD14 expression. Dendritic cells showed efficient phagocytosis and ability to stimulate T-lymphocytes. Moreover, dendritic cells could secrete high levels of interleukin-12 [IL-12] cytokine which was depictive of their full maturation. Measurement of the produced cytokines showed the generation of type-1 dendritic cells [DC1]. Our study showed that skin epithelial cells could induce maturation of monocyte-derived dendritic cells [DCs]. This feeder layer led to the production of efficient dendritic cells with the ability to be used for tumor immunotherapy

Study on the mechanism of polypeptide extract from scorpion venom to promote the restraint of cyclophosphamide on Lewis lung cancer / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the mechanism of polypeptide extract from scorpion venom (PESV) on promoting anti-tumor effects of cyclophosphamide (CTX).</p><p><b>METHODS</b>The Lewis lung tumor model was established by subcutaneously implanting Lewis lung cells into C57BL/6 mice. The tumor-bearing mice were randomly divided into 4 groups, i. e., the model group, the cyclophosphamide (CTX) group, the polypeptide extract from scorpion venom (PESV) group, and the combination group (CTX + PESV), 10 mice in each group. The tumor growth curve was recorded. Changes of vascular endothelial growth factor-A (VEGF-A) and transforming growth factor-beta1 (TGF-beta1) expressions in the tumor microenvironment were detected using reverse transcription PCR and immunohistochemical assay. Changes of dendritic cells (DCs) phenotype CD80 and CD86 expressions in the tumor tissue were detected using immunofluorescence chemical assay.</p><p><b>RESULTS</b>After 21 successive days of treatment, the growth of Lewis lung cancer transplantation tumor in the combination group was obviously inhibited (P<0.05). Compared with the model group,the expressions of CD80 and CD86 in the PESV group was somewhat enhanced, while those in the CTX group was somewhat lowered. Compared with the CTX group, the fluorescent signal strength and expressions in the combination group somewhat increased. Compared with the model group, the expressions of TGF-beta1 and VEGF-A mRNA decreased in the PESV group and the CTX group (both P<0.05). Compared with the PESV group and the CTX group, the expressions of TGF-beta1 and VEGF-A in the combination group both decreased (both P<0.05).</p><p><b>CONCLUSION</b>PESV could inhibit the expressions of VEGF and TGF-beta1, promote the maturation of DCs, recover its antigen uptake presentation function, and reverse the immune injury to the body by CTX, thus playing a role in inducing the tumor cell apoptosis.</p>

Involvement of dendritic cells frequency and function in outcomes of interferon-alpha therapy for chronic hepatitis B patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To elucidate the change in frequencies and functions of myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) before and after interferon-alpha therapy for chronic hepatitis B (CHB) patients, and its correlation with virological and biochemical data.</p><p><b>METHODS</b>Thirty patients with HBeAg-positive CHB who underwent IFN-alpha therapy were examined. Frequencies and expression of CD86 of mDC and pDC of peripheral blood were measured at baseline and treatment week (TW) 12 by flow cytometry. According to biochemical and virological parameters, the 30 patients were divided into ALT normalized group, ALT non-normalized group and virological responder group, virological non-responder group respectively. Statistical analysis of DC changes among different groups at baseline and TW12 was proceeded.</p><p><b>RESULTS</b>(1) In the ALT normalized group, the pDC frequency at TW12 (0.25 +/- 0.14%) was higher than that at baseline (0.18 +/- 0.09%) (P = 0.023); in the ALT non-normalized group, the mDC frequency (0.58 +/- 0.34%) and its surface CD86 expression (61.80 +/- 22.52%) decreased significantly as compared with baseline (0.88 +/- 0.51%, 79.92 +/- 25.94%, respectively), (P = 0.025, P = 0.036, respectively). (2) In the virological responder group, the CD86 expression on pDC at TW12 (46.86 +/- 12.22%) was higher than that at baseline (29.42 +/- 15.16%) (P = 0.002); in the virological non-responder group, the mDC frequency (0.51 +/- 0.22%) and its surface CD86 expression (59.63 +/- 22.94% ) decreased significantly as compared with baseline (0.94 +/- 0.58%, 80.11 +/- 29.34%, respectively), (P = 0.006; P = 0.049, respectively).</p><p><b>CONCLUSION</b>In IFN-alpha therapy for CHB patients, the increments of pDC frequency and function were related to biochemical and viral response, and decreases of mDC frequency and function were related to non-biochemical and non-viral response.</p>

Anti-tumor effect of cisplatin combined with DC vaccine on tumor-bearing mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the anti-tumor mechanism of the combination of cisplatin with DC vaccine in tumor-bearing mice.</p><p><b>METHODS</b>B16 melanoma cells were treated with cisplatin at the final concentration of 20 µg/ml in vitro for 24 h. The expression of HMGB1, Hsp70 and TGF-β were detected by Western blot. B16 tumor-bearing mouse models were generated. The therapeutic effect of the combination of cisplatin (100 µg/mouse i.p., for sequential 3 days) and intratumoral injection of DC cells (3×10(6)/mouse, twice with a 7-day interval) in the tumor-bearing mouse models was evaluated. Expression of MHC II, ICAM-1 and CD86 was analyzed by flow cytometry. The mice were sacrificed at 28 days after tumor cell inoculation. The tumors were removed and weighed, and tissue samples were taken for pathological examination. Tumor infiltrating lymphocytes (TIL) were isolated by discontinuous gradient centrifugation. The distribution of T-reg and CD8(+) T cells in the TIL was analyzed by flow cytometry, and the ratio of CD8(+) T/T-reg was determined. The activity of cytotoxic lymphocytes (CTL) was determined by microcytotoxicity assay.</p><p><b>RESULTS</b>Cisplatin enhanced both the B16 cell apoptosis and HMGB1 expression. After loading with cisplatin-treated cell lysate, the expression of MHC II, ICAM-1 and CD86 on DC cells were (47.5 ± 8.8)%, (35.5 ± 8.3)% and (36.2 ± 9.2)%, respectively. At 28 days after tumor cell inoculation, the tumor weight of the control group was (2.1 ± 0.6) g, that of the cisplatin group was (0.3 ± 0.2) g and that of cisplatin + DC vaccine group was (0.5 ± 0.2) g, showing a significant inhibition of tumor growth (P < 0.01). Furthermore, the CD8(+) T/T-reg ratio and CTL activity in TIL were also significantly enhanced in the tumor-bearing mice treated with cisplatin + DC vaccine. When the effector-to-target ratio was 20:1, 10:1 and 5:1, the CTL activity in the cisplatin + DC vaccine treated mice was (25.0 ± 5.0)%, (22.0 ± 6.0)% and (14.0 ± 4.0)%, respectively, significantly higher than (8.2 ± 3.6)%, (6.7 ± 1.8)% and (3.6 ± 1.9)%, respectively, in the control group (all P < 0.01).</p><p><b>CONCLUSION</b>Cisplatin promotes the anti-tumor effect of DC vaccine by down-regulating T-reg cells and enhancing the CTL activity in tumors.</p>

Expression of CD80 and CD86 on dendritic cells of patients with immune related pancytopenia and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the function of dendritic cells (DC) of patients with immune related pancytopenia (IRP) and explore the role of DC in IRP.</p><p><b>METHODS</b>The expression of CD80 and CD86 on myeloid DC (mDC, Lin-HLA-DR(+) CD11c(+) cells) and plasmacytoid DC (pDC, Lin-HLA-DR(+) CD123(+) cells) of 65 IRP (37 untreated and 28 remitted) patients and 17 healthy controls were analyzed by flow cytometry.</p><p><b>RESULTS</b>The expression of CD86 on pDC was (82.47 ± 13.17)% in untreated group and (60.08 ± 14.29)% in remission group, which were significantly higher than that of controls (47.95 ± 18.59)% (P < 0.05), while the expression in untreated group was higher than that of remission group (P < 0.05). The expression of CD80 on pDC was (6.31 ± 4.49)% in untreated group, which was significantly higher than that of remitted patients (3.09 ± 2.93)% and controls (2.33 ± 2.25)% (P < 0.05). The expression of CD86 on mDC was (97.06 ± 4.82)% in untreated group and (91.35 ± 12.20)% in control group, while the expression in untreated group was higher than that of control group (P < 0.05). The expression of CD80 on mDC was (6.20 ± 5.44)% in untreated group and (3.97 ± 3.24)% in remission group, which were significantly higher than that of controls (1.86 ± 1.73)% (P < 0.05). The expression of CD86 on pDC was negatively correlated to Th1/Th2 (r = -0.733, P < 0.05), it was positively correlated to the antibody on membrane of BMMNC (r = 0.283, P < 0.05) and the quantity of CD5(+)B cells (r = 0.436, P < 0.05), while it was negatively correlated to the level of hemoglobin, platelets and white blood cells (r = -0.539, P < 0.05; r = -0.519, P < 0.05; r = -0.567, P < 0.05, respectively). The expression of CD80 on pDC was negatively correlated to the level of hemoglobin and platelets (r = -0.431, P < 0.05; r = -0.464, P < 0.05).</p><p><b>CONCLUSION</b>The function of pDC in PB of IRP were strengthened, which was relevant to the immunopathogenesis of IRP.</p>

Human Leukocyte Antigen-G Expression on Dendritic Cells Induced by Transforming Growth Factor-beta1 and CD4[+] T Cells Proliferation

During antigen capture and processing, mature dendritic cells [DC] express large amounts of peptide-MHC complexes and accessory molecules on their surface. DC are antigen-presenting cells that have an important role in tolerance and autoimmunity. The transforming growth factor-beta 1 [TGF-beta1] cytokine has a regulatory role on the immune and non-immune cells. The aim of this study is to evaluate the effect of TGF-beta1 on the induction of human leukocyte antigen-G [HLA-G] expression on the DC which is derived from monocyte. Methods: In this study, we evaluated the effect of TGF-beta1 in induction HLA-G expression on the monocyte-derived DC by flowcytometry and then CD4[+] T cell proliferative responses in the presence of DC-treated TGF-[beta1] was studied. Results: The results of this study showed that DC bearing HLA-G down-regulated activation of CD4[+] T cells and production of IL-6 and IL-17 in comparison with control [P<0.05]. Conclusion: It is concluded that TGF-beta1 has an important regulatory role in CD4[+] T cell proliferation by increasing HLA-G on DC and these cells can probably prevent unexpected immune responses in vivo

Study on the functions of peripheral dendritic cells in chronic hepatitis B virus infection patients of Gan-depression Pi-deficiency syndrome and Gan-Dan damp-heat syndrome under different immune states / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the functions of peripheral dendritic cells (DCs) in chronic hepatitis B virus (HBV) infection patients of Gan-depression Pi-deficiency syndrome (GPS) and Gan-Dan damp-heat syndrome (GDS) under different immune states, thus to study the features of the immune expressions of the two syndromes in chronic HBV infection, providing objective evidence for Chinese medicine syndrome typing.</p><p><b>METHODS</b>The 40 chronic HBV patients were randomly assigned to two groups according to the immune state. Of them, there were 20 chronic HBV patients (under the condition of immune clearance; consisting of 10 patients of GPS and 10 of GDS) and 20 chronic HBV carriers (under the condition of immune tolerance; consisting of 10 patients of GPS and 10 of GDS). Besides, 10 healthy graduate volunteers of Hunan University of Traditional Chinese Medicine were recruited as the healthy control group. Their peripheral blood mononuclear cells (PBMCs) were cultured in vitro. The exterior morphological features and ultrastructure were observed by inverted microscope and electron microscope. The expressions of HLA-DR, CD80, CD86, and CDIa of the DCs surface were detected. The secretory levels of IL-12 in the supernate of DCs were detected by ELISA reagent kit. The proliferation capacities of allogeneic mixed lymphocyte were detected using MTT. The function features of DCs in the chronic HBV patients of two syndrome types under different immune states were compared, thus analyzing the difference of each index between the two syndrome types.</p><p><b>RESULTS</b>Compared with the healthy control group, the expression rates of CD86, CD80, and HLA-DR decreased in the HBV carriers group (of the two syndrome types), showing statistical difference (P < 0.05). The expression rate of CD80 decreased in the HBV group (of the two syndrome types), showing statistical difference (P < 0.05). The expression rates of CD86 and HLA-DR were lower in the GPS group than in the GDS group. The expression rate of CD80 was lower in the GPS group than in the GDS group, showing statistical difference (P < 0.05). The proliferation capacities of IL-12 and T lymphocytes were lower in the HBV patients group than in the healthy control group (P < 0.05). The proliferation capacities of IL-12 and T lymphocytes were lower in the GPS group than in the GDS group, showing statistical difference (P < 0.05).</p><p><b>CONCLUSIONS</b>The functions of peripheral DCs in chronic HBV infection of patients of the GPS and the GDS under different immune states were different. The phenotype and function tests of DCs provided objective evidence for Chinese syndrome typing of chronic hepatitis B, thus reflecting the features of immune expressions of the two syndrome types and the immunology connotation.</p>