Influence of allogeneic cornea on human peripheral blood T lymphocyte subtype in vitro

AIM: To observe the immunoregulation of allogeneic cornea on the human peripheral blood T lymphocytes in vitro. METHODS: After Co-culture of human peripheral blood lymphocytes and allogeneic cornea in vitro, T lymphocytes were labeled by monoclonal antibody, and analyzed by fluorescent activated cell sorter (FACS). RESULTS: CD25 expression on T lymphocytes in control was 25.2%, after stimulated by the allogeneic cornea or PDB, CD25 expression on T lymphocytes was 56.8% and 80.9%, respectively. After stimulated by the allogeneic cornea, CD25 expression on CD4+ or CD8+ T lymphocytes were 67.3% and 52.3%, respectively. CONCLUSION: Allageneic cornea stimulates CD25 expression on human peripheral blood T lymphocytes, and the CD25 expression on CD4+ T lymphocytes is more prominent than CD8+ T lymphocytes.     

Inhibitory effect of berberine on the activation and proliferation of T lymphocytes

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 μmol/L and 50 μmol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 μmol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G₀/G₁ phase to S and G₂/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.     

The inhibitory effect of quercetin on in vitro activation of T lymphocytes

AIM:To investigate the inhibitory effect of quercetin on in vitro activation of T lymphocytes by polyclonal activators with CD69 expression as an activation marker.METHODS:After being separated from lymphoid nodes of a C57BL/6 mouse, the lymphocytes were exposed to polyclonal activators (PDB or Con A) with or without quercetin. Then they were harvested at 2 h, 6 h and 24 h, respectively. The expressional rates of CD69 on T lymphocytes were assessed by two-color immunofluorescent staining and flow cytometry, and the inhibitory rates of quercetin at different time points were estimated.RESULTS:Quercetin had no effect on the expressional rate of CD69 on T lymphocytes under resting states. After the stimulation with PDB or Con A, the expressional rates of CD69 on T lymphocytes in the present of quercetin (10 μmol/L) showed significant decrease compared with those of control groups at different time points (P＜0.01). The inhibitory rate of quercetin on CD69 expression stimulated by PDB dropped sharply from 2 h to 24 h, whereas the inhibitory rate of quercetin on Con A action were relatively stable.CONCLUSION:Quercetin has inhibitory effects on the activation of T lymphocytes by Con A or PDB, suggesting that the action site of quercetin may be on PKCθ or its downstream. Furthermore,these inhibitory effect seems to be reversible.     

The characteristics of TCRVα24+ NKT cells in response to in vitro stimulation

AIM: To investigate the amount and patterns of expressing CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells, and compare with that of CD3⁺ T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin (Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells and CD3⁺ T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRVα24⁺ NKT cells to CD3⁺ T cells was about (1.34±0.42)%. The expression rates of CD69 on TCRVα24⁺ NKT cells and CD3⁺T cells in response to PDB+Ion for 6 h were (96.71±1.33)% and (98.60±0.47)%, respectively, while the ratio were (11.47±2.86)% and (1.07±0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-γ on TCRVα24⁺ NKT cells stimulated with PDB+Ion for 6 h were (48.62±2.44)% and (46.65±8.91)%, respectively, which were significantly higher than that of unstimulated group [(31.57±3.31)%, (13.45±6.29)%] and that of stimulated CD3⁺ T cells, though the expression rates on stimulated CD3⁺ T cells were significantly higher than that of unstimulated CD3⁺ T cells. CONCLUSION: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-γ and IL-4 on these lymphocytes are higher than CD3⁺ T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.     

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

AIM: To investigate the effect of dihydroartemisinin (DHA) on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS: Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester (CFDA-SE) staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3+ T lymphocytes, was measured by flow cytometry (FCM) combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration (［Ca2+］i) of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4+CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS: The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3+T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4+CD25high Treg. CONCLUSION: DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.     

Inhibitory effects of gossypol on the activation of human T-lymphocytes stimulated with polyclonal activators

AIM: Peripheral blood mononuclear cells (PBMC) were cultured in vitro to study the effect of gossypol, a polyphenolic antifertility agent, on the activation of normal human T cells. METHODS: Double fluorescent staining together with flow cytometry was adopted to analyze the influence of gossypol on expression of the early activation antigen CD69 on T-lymphocytes under stimulation of mitogen or phorbol ester. RESULTS:Analysis of T cell activation in vitro revealed that preincubation of PBMC with 100 μmol/L gossypol could completely inhibit the expression of early activation marker CD69 on CD3⁺ T cells in response to 10mg/L PHA, and block T cell activation by 10^-7 mol/L PDB as well. The suppression of CD69 expression was dose-dependent and IC₅₀ of gossypol on PDB and PHA were (35.7±2.9) μmol/L and (32.8±1.5) μmol/L(x ±s), respectively. Besides, gossypol had similar inhibitory effect on CD69 expression of CD3^- lymphocytes. However, it did not have any significant effect on T cell surface molecule CD3 down-regulation. CONCLUSION: Gossypol could inhibit T cell activation in vitro in response to polyclonal activators, both PHA and PDB, suggesting that its action site may be at PKC or its downstream and that gossypol possessed potential immuno-regulatory effect.     

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AIM: To study the effects of ultraviolet (UV) on mitochondrial functions and apoptosis in HaCaT cells. METHODS: After irradiation by UV at low dose (UVA 2 J/cm2，UVB 10 mJ/cm2) and high dose (UVA 6 J/cm2，UVB 30 mJ/cm2), HaCaT cells were cultured for 15 hours. Flow cytometry was used to measure mitochondrial membrane potential, mitochondrial mass and apoptotic rate. Annexin V-FITC/PI staining of apoptotic cells was analyzed by laser confocal microscopy. RESULTS: After UV irradiation, cell proportion with low mitochondrial membrane potential increased with irradiation doses. The proportion of control group, low dose group and high dose group were 7.94%±1.02%, 25.87%±4.55% and 39.27%±5.32%, respectively. Cells proportion with low mitochondrial mass increased with irradiation doses. The proportion of control group, low dose group and high dose group were 15.19%±1.58%, 40.36%±4.41% and 68.79%±5.46%, respectively. The hypodiploid peaks of DNA content analysis represented the apoptotic rate of HaCaT cells. The apoptotic rate of control group, low dose group and high dose group were 1.82%±0.51%, 30.16%±5.47% and 58.49%±5.98%, respectively. To analyze the cells apoptosis by staining with annexin V-FITC and PI, the results were consistent with those of DNA content analysis. Cells in control group showed almost no positive staining cells. Single annexin V-FITC positive cells in low dose group and double positive cells in high dose group were predominant, respectively. CONCLUSION: UV irradiation induces HaCaT cell mitochondrial depolarization, as well as mitochondrial mass loss. These changes are related to cell apoptosis.     

Effects of activated state of T cells from human peripheral blood on absorption of photosensitizer hematoporphyrin monomerthyl ether

AIM: To investigate the characterization of absorption of hematoporphyrin monomerthyl ether (HMME), a domestic new generation photosensitizer product, by activated T cells from human peripheral blood. METHODS: Evaluation was performed by flow cytometry on the effects of incubating concentration and time of HMME on absorption by activated T cells. Lymphocytes were separated from human peripheral blood by density gradient centrifugation with Ficoll and T cells were activated with polyclonal stimulators PHA and PDB+Ion. To analyze the effects of HMME incubating doses on the absorption of activated T cells, the cultural lymphocytes were incubated with a serial doses of HMME for 1 h and HMME absorption were measured by FACS after immuno-staining with anti-CD3 antibody. To test the impact of HMME incubating time on the absorption of activated T cells, the cultural lymphocytes were incubated with HMME for various times and HMME absorption were measured by FACS after immuno-staining with anti-CD3 antibody. RESULTS: The HMME absorption-dose curve and absorption-time curve were shifted to right and up in the activated T cells as compared to resting T cells. HMME absorptions of activated T cells were statistic significantly larger than that of resting T cells in the doses between 5 mg/L to 20 mg/L. HMME absorptions of either activated T cells or resting T cells underwent a gradual increase with the incubation-time in HMME at concentration of 10 mg/L. HMME absorptions of activated T cells were statistic significantly larger than that of resting T cells in the incubation-time between 15 to 60 min. CONCLUSION: The differences of HMME absorption between activated T cells and resting T cells depend on the incubation times and doses of HMME. HMME absorption of activated T cells are significantly larger than that of resting T cells in certain incubation-times and doses. These results suggest that incubation time and dose associated with HMME-PDT therapeutic windows will be created for selective deletion of activated T cells.     

Effect of isoflavone genistein on activation and proliferation of mouse T cells in vitro

AIM: To study the effect of genistein on activation and proliferation of T cells, and explore the molecular mechanism of genistein. METHODS: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin (ConA )and Phorbol 12, 13-dibutyrate(PDB) or T cell proliferation stained by CFSE in response to PDB / Ionomycin or ConA. RESULTS: Genistein inhibited the expression of CD69 and CD25 in activated T cells in response to Con A in a concentration-dependent manner and in response to PDB in a high concentration. Genistein inhibited proliferation of T cells in both groups in a concentration-dependent manner. CONCLUSION: Genistein inhibited activation and proliferation of T cells in vitro in response to polyclonal stimulus, and it may hold potential as a new immunosuppressant.     

Effects of psychological stress on in vitro expression of activated surface molecules on T cells of peripheral blood from healthy persons

AIM: To study cellular and molecular mechanism involved in increasing susceptibility of infection in psychological stress persons. METHODS: Comparative studies were performed with double staining and flow cytometry analysis on immunophenotyping and in vitro expression of early activating surface molecule CD69 in response to mitogens on T cells from peripheral blood of 20 healthy college student volunteers before and after psychological stress. A series of term final examinations was defined as psychological stress. RESULTS: Immunophenotyping analysis showed no statistically significant difference in the percentage of CD2, CD3, CD4, CD8, CD19, CD20, CD16 and CD56 positive lymphocyte populations before and after psychological stress. There was a statistically significant decrease in the in vitro expression of CD69 in response to polyclonal stimulators on the T cells from persons after psychological stress than those before psychological stress. The percentage of CD69 expression (CD69⁺CD3⁺/CD3⁺%) in response to PHA and PDB in the whole blood culture for 72 hours decreased respectively from 28.1±4.1 and 80.7±6.8 on the T cells obtained before psychological stress to 17.6±3.8 and 65.8±7.9 on those obtained after psychological stress, while there was no statistically significant difference between the CD69 expression rates without stimulators on the T cells obtained before and after psychological stress. CONCLUSIONS: The effects of psychological stress to immune system is not on the level of changing proportions of the sub-populations within peripheral blood lymphocytes. Psychological stress can decrease the activating response of T cells in healthy persons, which may be responsible for the increase of susceptibility to infection in the psychological stress persons.     

Correlation between CD4+CD28-T cells and lymphocytic apoptosis in rheumatoid arthritis patients

AIM: To investigate the fraction of CD4⁺CD28^-T cells and its correlation with lymphocytic apoptosis in peripheral blood of rheumatoid arthritis (RA) patients.METHODS: The RA patients and age-matched health controls were selected in the study. The lymphocytes were isolated from peripheral blood. CD4⁺ T cells without CD28 expression (CD4⁺ CD28^-) were analyzed by flow cytometry. The incidence of apoptosis in the cells cultured with or without PHA for 24 h was determined by flow cytometry with Annexin V-FITC/PI di-staining. The correlation between the fraction of CD4⁺CD28^-T cells and lymphocytic apoptosis was also observed.RESULTS: The fraction of CD4⁺CD28^-T cells was significantly higher in RA group than that in healthy control group (7.79%±3.52% vs 1.89%±1.78%, P<0.05). The apoptotic level in PHA cultured lymphocytes was significantly lower in RA group than that in healthy controls (11.38%±5.73% vs 19.46%±6.32％, P<0.05). Spearman analysis showed that there was a negative correlation between the fraction of CD4⁺CD28^-T cells and apoptotic level of activated lymphocytes (r=-0.433,P<0.01).CONCLUSION: The increased CD4⁺CD28^-T cells contribute to prolong the lifespan of activated lymphocytes in peripheral blood of RA patients, and the persistence of activated lymphocytes may contribute to the pathogenesis of RA.     

Effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro

AIM: To observe the effect of salidroside on behaviors of primary mouse T-lymphocytes in vitro. METHODS: The lymphocytes from the lymphoid nodes of BALB/c mice were isolated and primarily cultured. The viability of T cells was assessed by MTT assay. Fluorescence-conjugated monoclonal antibody and flow cytometry (FCM) were used to analyze the expression of T-cell activation marker CD69 in response to concanavalin A (Con A) in vitro. Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to detect the proliferation of T cells in vitro. FCM analysis was used to determine the production of reactive oxygen species (ROS) in the T cells by staining with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). The mean fluorescence intensity of DiOC₆(3) staining in the T cells was detected by FCM in order to analyze the effects of salidroside on the activity of the mitochondrial and the mitochondrial membrane potential in the T cells induced by dexamethasone (DEX). The thymus T cells from BALB/c mice were isolated and primarily cultured, and then FCM was also used to analyze the apoptosis of the thymus T cells treated with DEX. RESULTS: Salidroside increased the expression of T-cell activation marker CD69 at the final concentration of 80, 160 and 320 μmol/L (P<0.05). Salidroside promoted the proliferation of T cells induced by Con A for 72 h in vitro (P<0.01). Salidroside reduced the production of ROS (P<0.05) and protected the mitochondrial membrane potential of T cells from the injury of DEX (P<0.01). Salidroside also decreased the apoptosis rate of the thymus T cells induced by DEX in vitro (P<0.01). CONCLUSION: Salidroside promotes the activation and proliferation of T cells induced by Con A, reduces the production of ROS, maintains the mitochondrial membrane potential and protects thymus T cells against apoptosis induced by DEX in vitro.     

Synergistic effect of decitabine and valproic acid on apoptosis and cell cycle arrest at G0/G1 phase in gastric cancer MGC-803 cells

AIM: To investigate the synergistic effect of decitabine (DCA) and valproic acid (VPA) on apoptosis and cell cycle arrest at G₀/G₁ phase in gastric cancer MGC-803 cells. METHODS: Gastric cancer MGC-803 cells were used in the study and divided into the following groups according to the treatment with different drugs for 72 h: DCA 1.5 μmol/L,DCA 3.0 μmol/L, VPA 1.5 mmol/L, DCA 1.5 μmol/L+VPA 1.5 mmol/L and DCA 3.0 μmol/L+VPA 1.5 mmol/L. The early and late apoptotic rates were detected by annexin V and PI staining. The cell cycle was also determined by flow cytometry. The relative nm23-H1 mRNA expression level was measured by real-time quantitative PCR. RESULTS: The apoptotic rates in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (early: 33.58%±3.88%; late: 31.52%±4.20%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (early: 42.61%±4.23%; late: 38.01%±3.86%), the percentages of the cells in G₀/G₁ phase in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (61.55%±2.38%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (66.75%±2.48%), and the relative nm23-H1 mRNA expression levels in VPA 1.5 mmol/L +DCA 1.5 μmol/L group (1.84±0.46) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (3.02±0.36) were all significantly higher than those in the corresponding concentrations of single drug treatment groups (P<0.01). CONCLUSION: Synergistic effect of VPA and DCA on apoptosis and cell cycle arrest in gastric cancer MGC-803 cells is possibly via inactivation of nm23-H1 gene expression.     

Mechanisms underlying the induction of IL-2 secretion by PDB pl us ionomycin in CD4+CD25+ T cells from cord blood and adult peripheral blood

AIM:To confirm that CD4+CD25+ regulato ry T cells don't have an instinctive defection in IL-2 secretion,and to have an insight into the maturation state of CD4+CD25+ T cells in cord blood.METHODS:CD4+CD25+ and CD4+CD25- T cells were purified f rom cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS,and stimulated with PDB plus ionomycin.After 45 hours of culture,cells were d etected for expression of CD69 and CD25 by flow cytometry,and the supernatants were measured for 7 kinds of cytokines by Luminex.RESULTS:CD4+CD25+ T cells from both CB and PB proliferated comparably with CD4+CD25- T cells when stimulated with PDB plus ionomycin.A fter 45 hours of culture,however,the CD4+CD25+ T cells underwent a tendenc y of cell death.Expression of CD25 was further upregulated when CD25+ cells w ere activated.Under stimulation of PDB plus ionomycin,both CD4+CD25+ and C D4+CD25- T cells in PB secreted high levels of IFN-γ,IL-2 and TNF-α,with CD25+ cells secreted much higher level of IL-5,IL-4 and IL-10 than those in CD25- cells;CD4+CD25+ and CD4+CD25- T cells in CB also secreted high level of IL-2 and TNF-α but much lower level of IFN-γ than those in PB,and no secretion of IL-5,IL-4 and IL-10 was observed.CONCLUSION:CD4+CD25+ regulatory T cells don't have an i nstinctive defection in IL-2 secretion,otherwise there may be a different TCR signaling pattern in CD4+CD25+ T cells from traditional T cells.The CD4+C D25+ T cells in cord blood have not fully matured in function.     

Effects of oxymatrine on lymphocyte proliferation and the quantity of regulatory T cells

AIM: To analyze the effects of oxymatrine (OMT) on the quantity of murine regulatory T cells (Tr cells) in the peripheral blood and mouse lymphocyte proliferation stimulated by Con A, and to probe into the immunological mechanism that OMT treats allergic contact dermatitis (ACD).METHODS: An ACD mouse model stimulated by dinitrofluorobenzene (DNFB) was established. Different dosages of OMT, PBS and hydrocortisone (HCT) were intraperitoneally injected (IP) into the mice. Blood samples were collected at〖JP+2〗 1 d, 7 d, 14 d, 21 d and 28 d, then the T cells were isolated and marked with anti-CD3, anti-CD4, anti-CD25 three-colored immune fluorescence antibody to detect the quantity of CD4+CD25+ T cells with flow cytometry. The fluorescence intensity changes of lymphocytes which were isolated from mouses lymph node and co-stimulated by polyclonal stimulator Con A and OMT were examined by carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining and flow cytometry. RESULTS: OMT at concentrations of 500, 125 and 31 mg/L had the ability to restrain the proliferation of lymphocytes from lymph node in a dose dependent manner. However, OMT at concentrations of 16, 8, 4 and 2 mg/L promoted the proliferation of T lymphocytes from lymph node, but was not obviously dependent on its concentration. Intraperitoneal injection of OMT increased the numbers of CD4+CD25+T cell in peripheral blood obviously (P<0.01). CONCLUSION: The effects of OMT on the proliferation of T lymphocytes from mouses lymph node cells are observed, OMT also increases the CD4+CD25+T cells in the peripheral blood, implying that OMT is a kind of immunoregulator with dual effects.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

PIG7 and valproic acid promote differentiation and apoptosis of human leukemia SKNO-1 cells

AIM: To investigate the synergistic effect of p53-inducible gene 7 (PIG7) and histone deacetylase (HDAC) inhibitor valproic acid (VPA) on the differentiation and apoptosis of human leukemia SKNO-1 cells.METHODS: The DNA fragments containing PIG7 open reading frame or antisense oligonucleotides were subcloned into lentiviral vector. SKNO-1 cells were transduced with prepared lentivirus. Transgene expression was detected by semi-quantitative RT-PCR and Western blotting. The expression of myeloid cell differentiation antigen CD11b and the apoptotic cells were analyzed by flow cytometry. DNA fragmentation analysis was also used to observe the apoptosis of SKNO-1 cells.RESULTS: VPA inhibited the proliferation of SKNO-1 cells in a dose-　and time-dependent manner. Compared with control group, the differentiation and apoptosis of SKNO-1 cells were significantly induced by ectopically expressed PIG7 (P<0.05). The apoptosis induced by ectopically expressed PIG7 was further enhanced by VPA treatment (P<0.05), and the typical DNA ladders were also observed. The proportion of CD11b+ SKNO-1 cells notably increased after infection with lentivirus containing PIG7 as compared with empty vector group (P<0.05). Up-regulation of PIG7 also enhanced the susceptibility of the cells to the induction of differentiation by VPA.CONCLUSION: VPA inhibits the proliferation and induces the differentiation and apoptosis of SKNO-1 cells. Enforced expression of PIG7 enhances the differentiation and apoptosis of SKNO-1 cells and promotes the sensitivity of SKNO-1 cells to VPA. Over-expression of PIG7 combined with VPA may provide a new strategy for treatment of leukemia.     

Analysis of T cell activation and regulatory T cell derived from murine Peyer's patches

AIM: To explore the characteristics of T cell activation and regulatory T cells derived from murine Peyer's patches through comparative studies on Peyer's patches, mesenteric lymph nodes and inguinal lymph nodes. METHODS: Signal cell suspendsions were prepared from murine mesenteric lymph nodes (MLNs), the Peyer's patches (PPs) and inguinal lymph nodes (ILNs), respectively. The percentage of cell subpopulations such as CD3+ T cells, CD3+CD4+ helper T cells and regulatory T cells (Treg, CD4+CD25+) were analyzed. Lymphocytes were activated by polyclonal stimulators such as concanavalin (Con A), phorbol 12, 13-dibutyrate (PDB) only, and PDB plus ionomycin (Ion). The expression of CD69 (the early marker of CD3+ T cell activation) was measured by FACS. RESULTS: A lower ratio of CD3+ T cells was seen in PPs than those in MLNs and ILNs. The ratios of CD3+ CD4+ T cells to CD3+ T cells in PPs, MLNs and ILNs were almost the same. A higher rate of Treg was seen in CD4+ T cells from the PPs as compared with those from MLNs and ILNs. A higher percentage of activated CD3+ T cells derived from the PPs cultured without polyclonal stimulators were detected as compared to MLNs and ILNs, while lower responsiveness of CD3+ T cells from the PPs stimulated by Con A was seen as compared with those from MLNs and ILNs. CONCLUSIONS: The lower rate of CD3+ T cells as well as higher rate of Treg in PPs was due to its desensitization. The higher rate of basic activated state in CD3+ T cells from the PPs indicated that the T cells were activated by enteric antigens in physiological conditions. The lower responsiveness of activation to some polyclonal stimulators probably reveals that the T cells are in a state of anergy. All the characteristics mentioned above contribute to prevent pathological inflammations and maintain tolerance to enteric antigens such as food proteins and commensal bacteria but simultaneously retain proper immune responses to pathogenic microbes.     

Effect of TAP-SSL5 fusion protein on binding of activated platelets to human lymphocytes

AIM: To study the effect of tick anticoagulant peptide-staphylococcal superantigen like protein 5 (TAP-SSL5), an anti-inflammatory and anticoagulant fusion protein, on the binding of activated platelets to human lymphocytes.METHODS: Human periphery lymphocytes were isolated by magnetic activated cell sorting (MACS). The toxicity of TAP-SSL5 on the viability of Jurkat cell was assessed by CCK-8 assay. Flow cytometry was applied to detect the expression of CD162 (PSGL-1) on the Jurkat cells (human peripheral blood leukemia T lymphocyte cell line) and the inhibitory effect of TAP-SSL5 on the binding of mouse anti-human CD162 monoclonal antibody (KPL-1) to Jurkat cells. Platelets were activated by ADP at concentration of 20 μmol/L, the binding rates of activated platelets to Jurkat cells or human lymphocytes were assayed by flow cytometry. RESULTS: The concentration of TAP-SSL5 below 30 mg/L didn't affect the viability of Jurkat cells. TAP-SSL5 at 10 mg/L competitively inhibited KPL-1 binding to Jurkat cells. The binding rates of activated platelets to Jurkat cells or lymphocytes were (11.86±4.49)% and (8.32±1.00)%, respectively, which decreased to (6.73±2.71)% and (5.51±0.70)% after the Jurkat cells and lymphocytes were pre-incubated with 10 mg/L TAP-SSL5 (P <0.05).CONCLUSION: TAP-SSL5 binds to PSGL-1 expressed on lymphocyte surface and directly inhibits the binding of activated platelets to human lymphocytes, which may be one of the anti-inflammatory mechanisms of TAP-SSL5.