Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

Elevation of blood Th17 cells and CD3+CD8-IL-21+ T cells in patients with cervical cancer

AIM: To investigate the role of Th17 cells in the patients with cervical cancer.METHODS: We measured the peripheral levels of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in 37 cervical cancer (CC) patients, 25 cervical intraepithelial neoplasia (CIN) patients and 18 healthy controls by flow cytometry. The percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in total CD4⁺ cells were calculated.RESULTS: Compared with controls, the patients with CC or CIN had higher proportions of Th17 cells (all P<0.01) and CD3⁺CD8^-IL-21⁺ T cells (all P<0.05). Notably, in CC patients, the increased percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells were independently associated with the clinical stage(all P<0.05), lymph node metastasis (P<0.01,P<0.05) and vasoinvasion (all P<0.01), while the elevated percentage of CD3⁺CD8^-IL-21⁺ T cells was also associated with the tumor size(P<0.01). Furthermore, the percentage of Th17 cells was positively correlated with that of CD3⁺CD8^-IL-21⁺ T cells in healthy controls and CC patients, but not in CIN patients.CONCLUSION: Our results indicates a possible role of Th17 cells in CC patients correlated with CD3⁺CD8^-IL-21⁺T cells, and the elevated percentage of circulating Th17 cells may be involved in the development and progression of cervical cancer.     

Changes of CD28, CD56 and CD57 expression on CD8+T cells in the peripheral blood of healthy elderly individuals

AIM:The expression of CD28, CD56 and CD57 on CD8⁺T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS:Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8⁺CD28⁺, CD8⁺CD56⁺ and CD8⁺CD57⁺T cells in the peripheral blood between the young and elderly groups.RESULTS:CD8⁺CD28⁺T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07±5.28 and 49.84±7.43,respectively (P＜0.05). Conversely, CD8⁺CD56⁺T cells and CD8⁺CD57⁺T cells in the peripheral blood of the elderly group were significantly higher than those in the young group, and the percentage was 6.60±2.40 vs 2.10±0.35,41.82±6.01 vs 22.89±2.80, respectively(P＜0.05).CONCLUSION:The expression of CD28, CD56 and CD57 on the CD8⁺T cells in the peripheral blood are changed significantly with aging. The decrease in CD28 expression may play an important role in the immunosenescence, while the increase in CD56 and CD57 expression seems to be a compensatory adaptation for the immune dysfunction.     

CD4+CD25+FOXP3+ Tregs and HBV-specific CTLs in peripheral blood from chronic hepatitis B patients

AIM: To investigate the roles of CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) and HBV-specific cytotoxic T-lymphocytes (CTLs) in peripheral blood from the patients with chronic hepatitis B (CHB).METHODS: Peripheral blood mononuclear cells from 28 patients with CHB and 15 healthy controls were analyzed for Treg frequency using flow cytometry and for HBV-specific CTLs using enzyme-linked immunospot assay (ELISPOT).The clinical data of HBV-infected patients were considered.RESULTS: The frequency of CD4⁺CD25⁺FOXP3⁺Tregs was higher in the patients with CHB than that in the patients of healthy controls (3.14%±0.97% vs 1.95%±0.68%, P<0.05), and a positive correlation was found between Tregs and the DNA levels of HBV (r=0.831, P<0.01).HBV-specific CTLs were detected by ELISPOT in CHB patients and a negative correlation was observed between Tregs and CTLs (r=-0.540, P<0.01).CONCLUSION: Peripheral blood CD4⁺CD25⁺FOXP3⁺ Tregs in CHB patients are increased and closely correlated with the DNA replication of HBV and CTLs, suggesting that the clearance of HBV can be influenced by the inhibition of cellular immunoreaction through Tregs.     

Establishment of method for PD-1 immunophenotype detection in TCR Vβ repertoire of CD3+, CD4+ and CD8+ T-cell subsets

AIM:To establish the method for detecting the immunophenotype of immunosuppressive receptor programmed cell death protein 1 (PD-1) in T-cell receptor (TCR) Vβ repertoire of CD3⁺, CD4⁺ and CD8⁺ T-cell subsets, therefore to evaluate the distribution of PD-1 in T-cell repertoire from human peripheral blood (PB). METHODS:The PB samples from 10 cases of healthy individuals (HI) were collected. Using multi-colored fluorescence flow cytometry, the distribution frequency of PD-1 in TCR Vβ repertoire was detected with a wide panel of anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and 24 anti-TCR Vβ repertoire (IOTest^® Beta Mark TCR Vβ Repertoire Kit, containing 8 tubes which labeled A~H, each tube is a composite antibody of FITC and PE coupling, each cocktail contains antibodies direc-ted to 3 different Vβ subfamilies) monoclonal antibodies. RESULTS:The total number of the 24 TCR Vβ repertoire detected in CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells from 10 cases of HI was consistent with the Quick Reference Card data provided by the kit. The preliminary results showed that the frequency of Vβ usage in CD3⁺, CD4⁺ and CD8⁺ T cells was different. High usage of Vβ2, Vβ3, Vβ8, Vβ9, Vβ5.1, Vβ13.1 and Vβ13.2 was found in CD3⁺ T cells, while high usage of Vβ2, Vβ3, Vβ8, Vβ5.1, Vβ9 and Vβ13.1 in CD3⁺CD4⁺ T cells, and high usage of Vβ1, Vβ2, Vβ3, Vβ9, Vβ13.1 and Vβ13.2 in CD3⁺CD8⁺ T cells were also observed. Further analysis showed that the expression of PD-1 was detected in all 24 TCR Vβ subfamilies of CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. The higher frequency of PD-1⁺ T cells was CD4⁺Vβ5.2⁺ T cells, whereas the higher frequency of PD1⁺ T cells in CD8⁺Vβ11⁺ and CD8⁺Vβ13.6⁺ T cells was detected. CONCLUSION:The method for detection of the immunosuppressive receptor PD-1 in TCR Vβ repertoire of T-cell subsets is successfully established, which provides a new method for further analysis of immunosuppressive characteristics of TCR Vβ repertoire in the patients with leukemia.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.     

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.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Effect of pachyman polysaccharides on Th17/Treg balance in systemic lupus erythematosus patients

AIM: To investigate the immunomodulatory effect of pachyman polysaccharides (PPS) on T helper 17 cell (Th17)/regulatory T cell (Treg) balance in the peripheral blood of systemic lupus erythematosus (SLE) patients. METHODS: The CD4⁺ T cells were isolated from the peripheral blood samples obtained from 45 SLE patients and 35 healthy controls enrolled in our study using magnetic bead separation method. The proportions of Th17 and Treg cells were measured by flow cytometry. The CD4⁺ T cells from SLE patients and healthy controls were treated with PPS. The cytoto-xicity of PPS was evaluated by detecting cell viability with MTT assay. The contents of interleukin-17 (IL-17), IL-6, IL-10 and transforming growth factor-β (TGF-β) were measured by ELISA. The expression of retinoid-related orphan receptor γt (RORγt) and forkhead box protein P3 (Foxp3) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: The Th17 cells were significantly elevated, while Treg cells were obviously decreased in the SLE patients compared with the healthy control group (P<0.05). Compare with control group, the contents of IL-17 and IL-6 were decreased, while the contents of IL-10 and TGF-β were increased (P<0.05). The expression of RORγt at mRNA and protein levels was down-regulated and the expression of Foxp3 was up-regulated (P<0.05). The ratio of Th17/Treg was decreased in 100 μg/L nontoxic PPS-treated CD4⁺ T cells isolated from the SLE patients (P<0.05). CONCLUSION: PPS treatment inhibits Th17 cells and elevates Treg cells in the CD4⁺ T cells isolated from SLE patients, which may have a therapeutic effect on SLE patients.     

Relationship of CTCs with CD4+/CD8+, NLR,TTV and tumor stage in patients with primary hepatocellular carcinoma

AIM: To explore the relationship of circulating tumor cells (CTCs) with CD4⁺/CD8⁺, neutrophil-to-lymphocyte ratio (NLR), total tumor volume (TTV) and tumor stage in the patients with primary hepatocellular carcinoma.METHODS: We selected 80 cases of histologically diagnosed primary hepatocellular carcinoma in the study. The method of CanPatrol^TM was used, which was developed by SurExam biotechnology company to identify CTCs in the blood. CD4⁺ T cells and CD8⁺ T cells were counted by flow cytometry. The patients were divided into 2 groups according to the levels of CD4⁺/CD8⁺ ratio, NLR and TTV. The patients were also divided into Ⅰ＋ Ⅱstage group and Ⅲ＋ Ⅳ stage group according to the seventh edition of TMN staging criteria.RESULTS: The numbers of peripheral blood CTCs and mesenchymal CTCs in high CD4⁺/CD8⁺ ratio group were significantly lower than those in low CD4⁺/CD8⁺ ratio group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in high TTV group were significantly higher than those in low TTV group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs and hybrid CTCs in I＋ II stage group were significantly lower than those in III＋ IV stage group (P < 0.05). The numbers of peripheral blood CTCs, mesenchymal CTCs, hybrid CTCs and epithelial CTCs between high NLR group and low NLR group had no statistical difference.CONCLUSION: CTCs exist in the peripheral blood of the patients with primary hepatocellular carcinoma. The peripheral blood CTCs have significant correlations with TTV, tumor stage and T-cell immunity.The worse cell immune function, the larger TTV and the later tumor stage a patient has, the more the peripheral blood CTCs are.     

Glucocorticoids enhance differentiation of central memory CD8+ T-lymphocytes in vitro

AIM:To investigate the effect of glucocorticoids, a kind of traditional immunosuppressive drug, on expanding central memory CD8⁺ T cells (CD8⁺ T_CM) and to provide a novel method of generating CD8⁺ T_CM for adoptive immunotherapy.METHODS:Healthy human peripheral blood mononuclear cells were isolated by density gradient centri-fugation. Naïve CD8⁺ T cells were further isolated with MACS microbeads and flow cytometry. After activated by cytokines, the cells were divided into experimental group and control group. Glucocorticoids at different concentrations and an identical volume of PBS were added. The phenotypic characteristics of T_CM in different groups were measured by flow cyto-metry at separate time points. Furthermore, the effect of glucocorticoids on CD8⁺ T cell expansion was further verified using CFSE assay and Annexin V staining.RESULTS:Glucocorticoids significantly increased the proportion of CD8⁺ T_CM in vitro. Glucocorticoids sustained CD8⁺ T cell expansion. Glucocorticoids had low toxicity for CD8⁺ T cells.CONCLUSION:Glucocorticoids effectively increase the number and proportion of CD8⁺ T_CM in vitro, which provides novel insights into the generation of human CD8⁺ T_CM and reveals a novel potential clinical application of glucocorticoids for immunotherapy.     

EP2A promotes human CD34+ cell homing in vitro but not proliferation

AIM: To investigate the role of prostaglandin E₂ receptor 2 agonist (EP₂A) in proliferation and homing of human CD34⁺ cells. METHODS: Bone marrow fluid and peripheral blood containing stem cells were collected from healthy donors mobilized by granulocyte colony-stimulating factor in our department. Human CD34⁺ cells were isolated by the method of magnetic-activated cell sorting microbeads. Bone marrow mononuclear cells were isolated by Ficoll-Paque centrifugation, and the bone marrow mesenchymal stem cells (BMMSC) were cultured with L-DMEM. Human CD34⁺ cells and BMMSC were divided into 4 groups, and treated with PGE₂ (as positive control), DMSO (as negative control), EP₂A and EP₂A+prostaglandin E₂ receptor 2 antagonist (EP₂AA), respectively. After exposed to the reagents, human CD34⁺ cell viability was measured by CCK-8 assay, the number of colonies was evaluated by colony-formation assay, the cell cycle distribution was analyzed by flow cytometry, and the protein expression of survivin, β-catenin and CXC chemokine receptor 4 (CXCR4) was detrmined by Western blot. Moreover, the concentration of stromal cell-derived factor-1α (SDF-1α) in the BMMSC was detected by ELISA. RESULTS: The cell viability and the colony number of human CD34⁺ cells in EP₂A group were not higher than those in negative control group. Furthermore, the proportion of human CD34⁺ cells treated with EP₂A in G₂/M phase was not elevated compared with negative control group. The protein expression of survivin and β-catenin did not up-regulated in human CD34⁺ cells exposed to EP₂A, but the protein expression of CXCR4 in human CD34⁺ cells and the concentration of SDF-1α in BMMSC were elevated. CONCLUSION: EP₂A promotes human CD34⁺ cell homing in vitro but not proliferation.     

中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛征文通知

"中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛"将于2012年10月在西安举行,即日起征集:①研究论文摘要,②有关园艺学进展的综述文章。经审查合格的摘要和综述将分别收入"中国园艺学会2012年学术年会论文摘要集"和"庆祝《园艺学报》创刊50周年—园艺学进展论坛专辑",并于会前以     

Effects of thymosin α1 on the development and maturation of thymocytes

AIM: To investigate the effect of Thymosin α₁ on the development and matutation of thymocytes. METHODS: The proportion of CD4⁺CD8⁺ thymocytes and the expression of smoothened (Smo) of the hedgehog (Hh)-signaling in CD4^-CD8^-thymocytes were examined to observe the effect of thymosin α₁ on thymocyte development and matutation. RESULTS: Flowcytometric analysis showed that thymosin α₁ showed activity at a low dose of 30 μg/kg, and 30 μg/kg thymosin α₁ accelerated the replenishment and maturation of thymocytes according to the expression of Smo of the Hh-signaling in CD4^-CD8^-thymocytes, the potent negative regulator of proliferative responses. CONCLUSION: Thymosin α₁ can accelerates thymocyte development from CD4^-CD8^- to CD4⁺CD8⁺.     

Expression of Foxp3+ Tregs and PD1 in gastric cancer tissues and their correlation with clinicopathological factors and prognosis

AIM: To investigate the expression of Foxp3⁺ regulatory T cells (Foxp3⁺ Tregs) and programmed death receptor 1 (PD1) in gastric cancer tissues and their association with clinicopathological factors and prognosis of the patients. The correlation between the 2 molecules was also analyzed at the same time. METHODS: The tumor sections from 111 gastric cancer patients were stained for Foxp3 and PD1 by the method of immunohistochemistry. The associations of the expression levels of these 2 molecules with clinicopathological factors involved in the disease progression and prognosis were statistically analyzed. The relationship of their expression was detected. RESULTS: Foxp3⁺ Tregs and PD1 were expressed in the gastric cancer tissues, and PD1 was expressed in the tumor infiltrating lymphocytes (TILs). The expression of Foxp3 and PD1 was correlated with lymph node metastasis, clinicopathological stage and prognosis of gastric cancer patients. The expression of these 2 determinants in the patients with lymph node metastasis and an advanced clinicopathological stage was distinctly higher (P <0.05). The patients with positive expression of the 2 indexes presented a lower overall survival rate and worse prognosis (P <0.05). A significantly positive correlation between the infiltration of Foxp3⁺ Tregs and the expression of PD1⁺ TILs was also observed (P <0.01).CONCLUSION: Foxp3⁺ Tregs and PD1⁺ TILs co-infiltrate in the gastric cancer tissues, which can be used as biological markers to predict the disease progression and prognosis.     

Analysis of activation of T cells from draining lymph node and peripheral blood in allotransplantation mice

AIM:To study the activation of T cells from local lymph node and peripheral blood early after allotransplantation.METHODS:Transplant of myocardio-tissue into mouse forearm subcutaneously was used as a model to analyze the expression of CD69 by T subpopulations from draining lymph node and peripheral blood by flow cytometry.RESULTS:The expression rates of CD69 by both CD4⁺T cells and CD8⁺T cells from the draining lymph node were raised (P＜0.01) 72 h after allotransplantation, and it was higher on CD8⁺T cells than on CD4⁺T cells (P＜0.01). No significant difference in CD69 expression was found on CD4⁺T and CD8⁺T cells from peripheral blood among the groups, topical complete Freund's adjuvant (CFA) and systemic cyclosporin(CsA) enhanced and inhibited expression of CD69 by both CD4⁺T cells and CD8⁺T cells after allotransplantation, respectively (P＜0.05 or P＜0.01).CONCLUSION:To detect the expression of CD69 by T cells from draining lymph node can keep insight to the allorecognition early after transplantation.     

Imperatorin increased sensitivity of lung cancer CD133+ cell subsets to gefitinib by down-regulating c-met expression

AIM: To investigate the role of imperatorin in reversing the resistance of the PC9 CD133⁺ cell subsets to gefitinib. METHODS: MTT assay was performed to evaluate the viability of PC9 cells treated with imperatorin and gefitinib. The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot. The percentage of CD133⁺ cell subsets population and the apoptotic rate of the PC9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry. RESULTS: The sensitivity of the PC9 CD133⁺ cell subsets to gefitinib was significantly lower than that of the PC9 CD133^- cell subsets. Treatment with gefitinib alone significantly inhibited the protein levels of EGFR/PI3K/AKT in the PC9 CD133^- cell subsets but not the PC9 CD133⁺ cell subsets. Treatment with gefitinib alone increased the percentage of CD133⁺ cell subsets population in the PC9 cells. However, combination of gefitinib with imperatorin significantly inhibited the enrichment of CD133⁺ cell subsets population. Imperatorin down-regulated c-met expression, suggesting the c-met was the target of imperatorin in the PC9 CD133⁺ cell subsets. The results of MTT assay, Western blot analysis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC9 CD133⁺ cell subsets.CONCLUSION: Imperatorin increases the sensitivity of lung cancer CD133⁺ cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib.     

Detection and clinical significance of myeloid-derived suppressor cells in peripheral blood of patients with Parkinson disease

AIM: To detect the myeloid-derived suppressor cells (MDSCs) in peripheral blood from the patients with Parkinson disease (PD) and its clinical significance. METHODS: The patients (n=80) diagnosed PD from January 2016 to March 2017 in our hospital and 20 healthy volunteers were selected as the subjects. According to the Hoehn-Yahr staging, 80 PD patients were staged, of whom 22 were I, 24 were Ⅱ, 20 were Ⅲ, 14 were IV, and 0 was V. Peripheral blood (5 mL) samples from the patients with PD and the healthy volunteers were collected and the mononuclear cells were isolated. The levels of CD14⁺CD11b⁺ cells and CD14^-CD11b⁺ cells in the peripheral blood were detected by flow cytometry. The two populations of the cells were sorted by magnetic beads. The mRNA levels of arginase 1 (ARG1), interleukin-10 (IL-10) and cyclooxygenase 2 (COX-2) were detected by qPCR. The expression of surface membrane proteins CD14 and CD11b, and immunosuppressive factors ARG1, IL-10 and COX-2 was determined by Western blot and ELISA. RESULTS: No significant change of CD14⁺CD11b⁺ cells between the patients with PD and normal controls was observed, but the cells with CD14^-CD11b⁺ increased significantly in the patients with PD compared with the control people (P<0.05). The CD14^-CD11b⁺ cells in peripheral blood of the patients were related to the stage of Hoehn-Yahr. The CD14^-CD11b⁺ and CD14⁺CD11b⁺ cells showed high levels of IL-10 and COX-2, and the high level of ARG1 was only expressed in the CD14^-CD11b⁺ cells. The expression of ARG1 in the CD14^-CD11b⁺ population from PD patients was significantly different from that of CD14⁺CD11b⁺ population and normal subjects (P<0.05). CONCLUSION: The CD14^-CD11b⁺ cells and ARG1 expression level in peripheral blood of the PD patients can be used to evaluate the pathogenesis and staging. Immunosuppression may play an important role in the pathogenesis and development of PD.