Extrinsic Acquisition of CD80 by Antigen-Specific CD8⁺ T Cells Regulates Their Recall Immune Responses to Acute Viral Infection

CD80 is mainly expressed on Ag-presenting cells (APCs) as a costimulatory molecule but is also detected on T cells. However, the origin and physiological role of CD80 on CD8⁺ T cells remain unclear. In the present study, we demonstrated that effector and memory CD8⁺ T cells, but not naïve CD8⁺ T cells, displayed CD80 molecules on their surfaces after acute lymphocytic choriomeningitis virus infection. Using adoptive transfer of CD80-knockout (KO) CD8⁺ T cells into a wild type or CD80-KO recipient, we demonstrated that the effector CD8⁺ T cells displayed CD80 by both intrinsic expression and extrinsic acquisition, while memory CD8⁺ T cells displayed CD80 only by extrinsic acquisition. Interestingly, the extrinsic acquisition of CD80 by CD8⁺ T cells was observed only in the lymphoid organs but not in the periphery, indicating the trogocytosis of CD80 molecules via interaction between CD8⁺ T cells and APCs. We compared the recall immune responses by memory CD8⁺ T cells that either extrinsically acquired CD80 or were deficient in CD80, and found that CD80, presented by memory CD8⁺ T cells, played a role in limiting their expansion and IL-2 production upon exposure to secondary challenge. Our study presents the in vivo dynamics of the extrinsic acquisition of CD80 by Ag-specific CD8⁺ T cells and its role in the regulation of recall immune responses in memory CD8+ T cells.

Changes of DC Subsets and CD80 and CD86 Expression in Peri-pheral Blood of Patients with ITP and Their Correlation with Efficacy of Dexamethasone Treatment / 中国实验血液学杂志

OBJECTIVE@#To analyze the changes of DC subsets and the expression of CD80 and CD86 in peripheral blood of ITP patients and their correlation with dexamethasone efficacy.@*METHODS@#Peripheral blood sample of 80 cases of ITP and 20 normal controls from June 2015 to June 2017 in our hospital were retrospectively analyzed. The specific distribution of DC subsets in the peripheral blood of all the subjects was detected by flow cytometry, and the expressions of CD80 and CD86 were detected by ELISA.@*RESULTS@#The proportion of DC2 in DC subsets of ITP patients before treatment was significantly higher than that in normal control group (P<0.05). The proportion of DC2 in DC subset of ITP patients was still significantly higher than that of the control group (P<0.05). The level of CD80 expression on DC1 and DC2 in ITP patients before treatment was significantly higher than that in the normal control group (P<0.05), and the expression level of CD86 on DC2 was significantly higher than that of the normal control group (P<0.05). Both IL-2 and IFN- γ levels in the patients before the treatment were significantly higher than those in the normal control group (P<0.05), and the expression levels after treatment with dexamethasone decreased significantly. Before treatment, both IL-4 and IL-10 levels in ITP patients were significantly lower than those in the normal control group (P<0.05), and their expression levels after treatment with dexamethasone significantly increased (P<0.05).@*CONCLUSION@#The incidence of ITP patients closely relates with the level and dysfunction of DC subsets in peripheral blood and the expression levels of IL-2, IL-4, IL-10, IFN- γ, which significantly correlates with the efficacy of dexamethasone.

Adiponectin inhibits oxidization-induced differentiation of T helper cells through inhibiting costimulatory CD40 and CD80

Adiponectin is a multifunctional adipokine that has several oligomeric forms in the blood stream, which broadly regulates innate and acquired immunity. Therefore, in this study, we aimed to observe the differentiation of T helper (Th) cells and expression of costimulatory signaling molecules affected by adiponectin. The mRNA and protein expression levels of adiponectin and its receptors in oxidized low density lipoprotein cholesterol-treated endothelial cells were assayed by real time PCR and immunofluorescence. The endothelial cells were then treated with adiponectin with or without adipoR1 or adipoR2 siRNA and co-cultured with T lymphocytes. The distribution of Th1, Th2 and Th17 subsets were assayed by flow cytometry. The effects of adiponectin on costimulatory signaling molecules HLA-DR, CD80, CD86 and CD 40 was also assayed by flow cytometry. The results showed that endothelial cells expressed adiponectin and its receptor adipoR1 and adipoR2, but not T-cadherin. Adiponectin suppressed Th1 and Th17 differentiation through adipoR1 receptor, contributed to the inhibition of CD80 and CD40, and inhibited differentiation of Th1 and Th17 by inhibiting antigen presenting action.

New insight in pathogenesis of podocyte disfunction in minimal change disease / 浙江大学学报·医学版

Minimal change disease (MCD) is a common pathological type of nephrotic syndrome. Its main histology is the fusion of podocyte foot process. The pathogenesis of MCD is not clear, but previously it was thought to be related to immune mechanism. In recent years more studies show that podocyte injury is the key link in the pathogenesis of MCD. In MCD mouse model and human kidney tissues, the expressions of podocyte slit membrane protein-nephrin and podocin, skeleton protein-synaptopodin are decreased, and the expression of synaptopodin is correlated with the response to hormone therapy. In addition, newest studies focused on another two potocyte associated proteins, CD80 and Angiopoietin-like-4. CD80, a T cell stimulating molecule, is expressed in potocyte. Kappa B gene sequences can be activated by external microbes, antigens through acting potocytes, which can induce the upregulation of CD80 expression, cytoskeletal protein damage and the glomerular filtration rate changes, resulting in proteinuria. Angiopoietin-like-4 can be expressed in normal potocytes, but over-expression of angiopoietin-like-4 may injure the GBM charge barrier and induce the foot process fusion, leading to MCD. However, further studies on the factors inducing CD80 and Angiopoietin-like-4 expression, and the interaction between glomerular basement membrane and the two proteins are needed. Based on the mechanism of MCD, NF-kappa B inhibitors and sialylation therapy would be a novel non-immune therapy for MCD.

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>

Cohort Study on GHA and New Combined Priming Chemotherapeutic Regimens in Treatment of Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical efficacy and adverse effects of GHA(G-CSF+homoharringtonin+cytarabine C) and new combined priming chemotherapeutic regimens(GHAA/GHTA) with high efficacy and low toxicity for treatment of relapsed and refractory acute myeloid leukemia(AML) and myelodysplastic syndrome(MDS), and to analyze the relation of above-mentioned regimens with the expression of co-stimuolating molecule B7.1.</p><p><b>METHODS</b>Standard GHA regimen consisting of G-CSF: 100 µg/(m2·d) subcutaneous injection, d 0-14; homoharringtonine: 1.0 mg/(m2·d) intravenous drip, d 1-14; Ara-C: 7.5-10 mg/(m2·d) subcutaneous injection, q12h, d 1-14. Other regimens as GHAA/GHTA were combined respectively with aclarubicin 20 mg d 1-7, or pirarubicin 20 mg d 1-7. 74 patients with refractory AML and 46 patients with MDS received these priming chemotherapy. The clinical efficacy and toxicity of above-mentioned priming chemotherapy were compared with 56 patients received routine chemotherapy (MA/TAE) respectively. And the expression of costimulatory molecule B7.1 on leukemia cells in patients of different subtypes was also detected by immunofluoressence and its relationship with clinical efficiency was explored.</p><p><b>RESULTS</b>(1) for AML patients treated with priming chemotherapy, the total remission was 67.56% (CR 54.05%, PR 13.51%), which was much higher than that of patients received routine chemotherapy (P<0.05). The CR rate of AML-M2 and AML-M5 group (65.51%, 61.90% respectively) was much higher than that of AML other subtypes (P<0.05), and the longest remission period lasted for 4 years; (2) for MDS patients treated with priming chemotherapy, the total remission was 60.87% (CR 45.65%, PR 15.22%), which was also significantly higher than that of patients received routine chemotherapy (P<0.05); (3) in comparison with patients received standard GHA priming regimen, the remission rate of combined priming chemotherapy GHAA/GHTA was significantly higher both in patients with AML (85.18%) and MDS (81.25%); (4) side effects after chemotheropy, including granulocyte deficiency, thrombocytopenia and anemia etc, lasted for 7-14 days; the severe infection rate was 1%, there were no severe bleeding, digest effect and damage of function in heart, liver and kidney. The therapy-related mortality was zero. Compared with routine chemotherapy, priming chemotherapy proved significantly safe and effective (P<0.05); (5) the expression rate of costimulatory molecule B7.1 showed large variance between AML and MDS, it was higher in AML-M2/AML-M5 and lower in AML of other subtypes (P<0.05). In the same case, B7.1 expression was positive correlated with efficiency of priming chemotherapy.</p><p><b>CONCLUSION</b>GHA priming chemotherapy, as well as other combination regimens GHAA/GHTA, are well-tolerated, effective regimens for refractory AML and advanced MDS, without severe side effects and therapy-related mortality. Especially the new regimens GHAA/GHTA has better efficacy, which are proved more efficient than conventional GHA. Efficiency of priming chemotherapy is positive correlated with B7.1 expression, its mechanism will be further explored.</p>

Influence of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice / 中国实验血液学杂志

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.

Experimental study on anti-tumor effect of xihuang pill and its immune clearance function / 中国中药杂志

<p><b>OBJECTIVE</b>To discuss the anti-tumor effect of Xihuang pill on tumor-bearing rats and its effect on the immune clearance function of tumor-bearing organisms.</p><p><b>METHOD</b>Walker256 tumor cells were adopted to establish the tumor-bearing rat model. The rats were randomly divided into five groups: the normal control group, the model control group, the lentinan group and Xihuang pill low dose, middle dose and high dose groups, with 10 rats in each group, and continuously treated and given drugs for 14 d after modeling. Blood and tumors were collected from abdominal aorta to calculate the tumor inhibition rate. The content of CD3+, CD4+, CD8+ T cells and adhesion molecule B7-1 (CD80) in peripheral blood were detected by flow cytometry (FCM). The expressions of IL-2 and IFN-gamma in were determined by ELISA.</p><p><b>RESULT</b>The tumor inhibition rate of the Xihuang pill high dose group was 33. 1 percent. Compared with the model group, the Xihuang pill large dose group showed significantly low IL-2, IFN-gamma, CD3+, CD4+, B7-1 in peripheral blood, with statistical significance in their differences (P < 0.05).</p><p><b>CONCLUSION</b>Xihuang pill could show its anti-tumor effect by enhancing the immune clearance function and increasing IL-2, IFN-gamma, CD3+ T, CD4+ T, B7-1 in peripheral blood.</p>

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.

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.

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

Role of miR-155 in myasthenia gravis and effect of dexamethasone on miR-155 / 中南大学学报(医学版)

OBJECTIVE@#To determine the role of miR-155 in the pathogenesis of generalized myasthenia gravis (GMG) and the effect of dexamethasone (DXM) on miR-155.@*METHODS@#The expression of miR-155 in B cells from the GMG patients and healthy controls was analyzed by qPCR. The B cells were cultured with DXM and PBS. The B cell proliferation was examined by MTT; CD80 and CD86 frequencies were detected by flow cytometry; and anti-AChRIgG and isotypes anti-AChR-IgG1, 2, 3 in the supernatant were detected by ELISA.@*RESULTS@#qPCR revealed that the expression of miR-155 in the B cells was much higher than that in the controls, and the miR155 expression decreased after DXM treatment. flow cytometry showed that there was no significant difference in the proliferation and the expressions of CD80 and CD86 in the B cells between the DXM group and the PBS group. The concentration of anti-AChR-IgG1 was obviously lower in the DXM group than in the PBS group, but the concentration of anti-AChRIgG, anti-AChR-IgG2, and anti-AchR-IgG3 was similar.@*CONCLUSION@#high expression of miR-155 may be associated with myasthenia gravis progression. DXM may disturb the antibody class switch of B cells by suppressing the expression of miR-155 and improve the symptom of MG patients.

Construction of an anti-tumor immunogene vaccine pVAX-IL-12-GB and its expression in vivo and in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.</p><p><b>METHODS</b>Human IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1, and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000. The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA, and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay. The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation, and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.</p><p><b>RESULTS</b>Enzyme digestion, PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB. IL-12, GM-CSF and B7.1 expressions were all detected in transfected 293T cells, and the expression of IL-12 was also detected in the transfected mouse muscular tissues.</p><p><b>CONCLUSION</b>A novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro, which facilitates further studies of tumor immunogene therapy.</p>

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>

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>

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>

Comparing effects of interleukin-2 and interleukin-4 on the expression of MHC class II, CD80 and CD86 on polymorphonuclear neutrophils

Polymorphonuclear neutrophils [PMN] have contributed only to the innate immune response, due to their phagocytosis activity. They have a short life time. Some studies of PMN cytokine production and expression of numerous cell surface proteins have suggested that PMN are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Flow cytometry was used in the present study for the detection of cell surface major histocompatibility complex [MHC] class II, CD80 and CD86 required for antigen presentation and subsequent T-cell activation. Human peripheral blood neutrophils were used for this purpose. In vitro stimulation of PMN with IL-2 or IL-4 for 24 h showed expression of MHC class II, CD80 and CD86. These observations, therefore, provide support to the hypothesis that human PMN have the potential to express molecules required for antigen presentation