Effects of peripheral-type benzodiazepine receptors in platelet membrane on patients with depression after first attack of cerebral infarction

AIM：To evaluate the changes of peripheral-type benzodiazepine receptors (PBRs) in platelet membrane in post-stroke depression (PSD) patients and to investigate the effects of PBRs on PSD. METHODS：Forty-three patients with PSD, fifty-nine patients with first-ever cerebral infarction and fifty-six healthy volunteers participated in this study. Platelet membrane in venous blood was prepared. Binding assay of the radioactive PBRs antagonist ［3H］PK11195 to platelet membrane was performed. RESULTS：A significant difference of ［3H］PK11195 binding was found among the 3 groups (P<0.01). Compared with the healthy volunteers ［(298.2±25.1） pmol/(g protein)］, a highly significant increase in ［3H］PK11195 binding was observed in platelet membrane in the patients with first attack of cerebral infarction ［(1 410.8±41.4） pmol/(g protein), P<0.01］. Compared with the patients with first attack of cerebral infarction, a significant reduction in ［3H］PK11195 binding was detected in platelet membrane in the patients with PSD ［(361.7±30.6) pmol/(g protein), P<0.01］. In the patients with PSD, no significant difference of ［3H］PK11195 binding was found between men and women (P>0.05). ［3H］PK11195 binding was related to the score of Hamilton depression rating scale (r=-0.44, P<0.01) but not to the duration of cerebral infarction (r=0.27,P>0.05). CONCLUSION:PBRs binding activity in platelet membrane decreases in the patients with PSD and affects the degree of depression.     

Assessment of affecting factors in measuring activating platelets with the method of flow cytometry

AIM:To investigate the affecting factors of detecting platelet activation by flow cytometry (FCM).METHODS:Using decoagulant of natrium citricum, anticoagnlated peripheral venous bloods from 6 healthy donors were labeled with the method of three-colour immunofluorescence assay.Platelet activation markers fibrinogen receptor (Fib-R, PAC-1) and P-selectin (CD62P) were measured.In the same time, the reproducibility of FCM was assessed.RESULTS:The platelet activation markers PAC-1 and CD62P at each time point showed significant difference(P<0.05).The ratio was increased with time extending.The positive ratio of PAC-1 and CD62P immediately measured (within 10 min) was 2.7% and 3.5% less than those at time point of 30 min.The results were measured several times under different activation levels.The coefficient of variation was less than 5%.CONCLUSION:In room temperature and with decoagulant of natrium citricum, if the measurements of PAC-1 and CD62P are finished within 30 min after sampling, good reproducibility should be achieved.     

Association between apolipoprotein E polymorphism and lipid metabolism in patients with cerebral infarction

AIM:To study the relationship between ApoE polymorphism and lipid metabolism of patients with cerebral infarction. METHODS:ApoE phenotype was determined in 110 patients with cerebral infarction and 60 normal controls by isoelectric focusing and immunoblotting. The TC, TG and HDL-C levels in serum of these subjects were measured with enzymes methods, ApoA I and ApoB levels with rocket immunoelectrophoresis methods, ApoE and Lp(a) levels with ELISA methods. RESULTS:The differences of the ApoE polymorphism distribution and ApoE allele frequencies (P＜0.05) occurred between two groups. The frequence of ApoE ε4 allele in patients with cerebral infarction was significantly higher than those in the normal controls (P＜0.05). However, ApoE 3/3 phenotypes was significantly lower (P＜0.05). Comparison of values of serum lipid with various ApoE phenotyoe among patients with cerebral infarction revealed that there was correlation between ApoE polymorphism and TC(P＜0.05), TG(P＜0.05), HDL-C(P＜0.05), LDL-C(P＜0.05), ApoA I(P＜0.05), ApoB(P＜0.05), ApoE(P＜0.05)and Lp(a)(P＜0.05). Patients carrying ε4 were associated with increased TC, LDL-C, ApoB and Lp(a), while those with ε2 were assiociated with decreased TC, LDL-C, and ApoB. Patients carrying ε2 were associated with increased TG, HDL-C, ApoAⅠ, and ApoE.CONCLUSIONS:ApoEε4 allele was associated with the development of cerebral infarction. ApoE polyphorphism affects lipid metabolism of cerebral infarction patients.     

水杨酸和高温锻炼与葡萄抗热性及抗氧化的关系

 对‘京秀’葡萄(Vitis vinifera CV．Jingxiu)高温锻炼和外施水杨酸(salicylic acid，SA)都能显著提高其幼苗的抗热性。高温锻炼1 h，叶片内自由态sA含量急剧升高，其后又迅速下降。在抗热性诱导过程中(高温锻炼12 h或水杨酸喷施后6 h)，抗氧化酶系统中的过氧化物酶(POD)、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)、抗坏血酸过氧化物酶(APX)的活性都明显升高，但过氧化氢酶(CAT)活性下降；高温锻炼或外施SA 1 h，过氧化氢(H2o2)含量急剧升高，之后又迅速下降，可能起着一种信号分子的作用。以上结果说明内源sA可能通过提高抗氧化酶活性参与了高温锻炼过程，外施sA和高温锻炼有相似的提高抗热性机制。     

Effects of pretreatment with captopril on the infarct size and myocardial cell apoptosis in experimental rabbits

AIM:To investigate the effects of pretreatment of captopril on the infarct size and myocardial cell apoptosis in rabbits. METHODS:Rabbits were randomly divided into sham-operated control group (SO), acute infarct group (AI) and captopril pretreatment group (CP). The rabbits of CP group were treated with captopril (25 mg·kg^-1.d^-1) for 1 week before harvest. The left circumflex branch of coronary (LCX) was ligated to develop acute ischemic model. The systolic and diastolic function of left ventricle(LV) was measured before and at 15, 30, 60 min after ligating LCX, and the blood viscosity and hematocrit before and at 60 min after ligating LCX were measured also. 6 hours later LCX ligation, the hearts were harvested for determining the infarction size, which was expressed as the ratio of infarct area to the total ischemic area, and evaluating apoptosis index expressed as the percentage of myocardial cells with TUNEL positive staining. RESULTS:1.Compared with AI group, captopril pretreatment significantly reduced the infarction size (16.60%±0.94% vs 36.24%±1.94%, P＜0.05), and improved the LV function and viscosity of blood. 2. Apoptosis of myocardial cell was found in the myocardium surrounding to the infarction area, however, the apoptosis index of CP group was significantly lower than that of AI group (26.30%±0.71% vs 42.44%±2.32%,P＜0.05).CONCLUSION:Apoptosis of myocardial cell exists in the area surrounding the infarction. Captopril pretreatment can reduce infarction size and myocardial apoptosis index, and improve the LV function as well as blood viscosity in this acute ischemic model.     

Correlation between clinical stages of HBV chronic infection and co-expression of CD244 and PD-1 in HBV-specific CTLs

AIM: To investigate the co-expression of cluster of differentiation 244 (CD244) and programmed death 1 protein (PD-1) on hepatitis B virus (HBV) antigen-specific cytotoxic T-lymphocytes (CTLs) in chronic hepatitis B (CHB) patients and its correlation with the clinical stages of HBV chronic infection. METHODS: Eighty-one CHB subjects with human leukocyte antigen-A2 (HLA-A2) positive, including 20 cases of acute-on-chronic liver failure (ACLF), 20 cases of severe chronic hepatitis B (s-CHB), 34 cases of mild and moderate chronic hepatitis B (m-CHB) and 7 cases of immune tolerant stage (IT) of chronic hepatitis B as well as 14 healthy controls (HC), were enrolled in this study. The co-expression of CD244 and PD-1 was analyzed in virus-specific CD8⁺ T-cells derived from peripheral blood using major histocompatibility complex class I (MHC-I) pentamers targeting immunodominant epitopes of HBV core antigen (18-27). RESULTS: In the patients with chronic HBV infection, virus-specific CD8+T-cells with co-expression of CD244 and PD-1 were at increased levels than those in total CD8+ T-cells (67.48%±17.16% vs 14.01%±7.97%, P<0.01) in the peripheral blood. Among different clinical stages, increased level of CD244 expression coincided with increased expression of PD-1 in m-CHB compared with IT of CHB (73.08%±8.63% vs 53.11%±18.05%, P<0.05), which was followed by decreased co-expression level in ACLF (63.11%±13.87% vs 72.05%±16.86%, P<0.05) and restoration of the ability to secrete IFN-γ (30.95%±20.29% vs 13.63%±10.46%, P<0.05). CONCLUSION: CD244 and PD-1 are highly co-expressed in HBV-specific CTLs in the patients with s-CHB and m-CHB, and are decreased in ACLF following the restoration of IFN-γ secretion. The severity of CHB may be correlated with CD244 and PD-1 co-expression in HBV-specific CTLs.     

Correlation analysis between visualization of hemorrheologic changes and platelet function in patients with acute coronary syndrome after percutaneous coronary intervention

AIM:To intuitionally observe the characteristics of blood rheology in the patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI) for 1 year to 3 years by micro-channel array flow analyzer (MC-FAN) combined with other platelet function indexes, and to explore the correlations between the test results of MC-FAN and platelet function. METHODS:This study brought 74 patients with ACS after PCI for 1 year to 3 years into test group, and 21 healthy subjects were enrolled as normal group. The levels of platelet aggregation test (PAgT), platelet adhesiveness test (PAdT), P-selectin, platelet-derived growth factor BB (PDGF-BB) and von Willebrand factor (vWF) were detected. MC-FAN HR300 was used to detect the transiting time (MC-FAN TT) of the blood passing through the model body capillaries. The differences of the test results between the 2 groups were compared, and the correlations between the results of MC-FAN and platelet function in the patients with ACS after PCI were also explored. RESULTS:Compared with normal group, the MC-FAN TT in test group was prolonged (P<0.01), the ability of erythrocyte deformation was weakened, and the leukocyte attaching the vascular wall and platelet adhesion and aggregation relatively increased. The levels of PAgT, PAdT, P-selectin and PDGF-BB in test group were all higher than those in normal group (P<0.01). No difference of vWF between the 2 groups was observed. The intergroup correlation analysis showed that there were correlations between MC-FAN TT and platelet function, in which 10 μL MC-FAN TT and 30 μL MC-FAN TT had the most significant correlation with P-selectin (r=0601, P<0.01; r=0334, P<0.01), 60 μL MC-FAN TT had the most significant correlation with PAgT (r=0527, P<0.01), and 100 μL MC-FAN TT had the most significant correlation with PAdT (r=0. 815, P<0.05). CONCLUSION:The visualization of hemorrheologic changes and platelet function in the patients with ACS after PCI are abnormal.There are correlations between MC-FAN TT and platelet function.The results of MC-FAN can objectively evaluate the blood rheology of the patients, and provide the reference for clinical treatment.     

Relationship between matrix metalloproteinase-9 and tissue metalloproteinase inhibitor-1 and carotid atheromatous plaque stability

AIM: To investigate the correlation between matrix metalloproteinase-9 (MMP-9),tissue metalloproteinase inhibitor-1 (TIMP-1),MMP-9/TIMP-1 and carotid atheromatous plaque stability in cerebral infarction patients.METHODS: 80 patients with cerebral infarction were categorized as microemboli-negative group (n=70) and microemboli-positive group (n=10),20 normal human were served as control group.The MMP-9 and TIMP-1 levels in plasma were determined by mean of ELISA in 3 groups.RESULTS: The levels of MMP-9 and TIMP-1 in plasma were significantly higher in cerebral infarction patients than those in control group (P<0.01).The plasma MMP-9 content was positively correlated with TIMP-1 content (r=0.76，P<0.01).The ratio of MMP-9/TIMP-1 increased only in microemboli-positive patients (P<0.01).CONCLUSION: The results indicate that the plasma MMP-9 participates in pathophysiological process of cerebral infarction.The ratio of MMP-9/TIMP-1 shows a close relationship with carotid atheromatous plaque instability.     

Balance of Treg/Th17 in synovium of collagen-induced arthritis rats and impact of TNF-α blockage therapy

AIM: To investigate the balance of Treg/Th17 in synovium of collagen-induced arthritis (CIA) and the impact of tumor necrosis factor α(TNF-α) blockage therapy. METHODS: Rat CIA model was established by bovine II collagen injection. The pathological score was evaluated by HE staining and toluidine blue staining. The TNF-α level in plasma was measured by ELISA. The expression of Treg/Th17 in synovium was detected by double staining immunofluorescence. RESULTS: The plasma level of TNF-α in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (P<0.01), whereas no significant difference was found between TNFR-Fc treatment group and control group (P>0.05). No significant difference between CIA group and control group in the ratio of CD4⁺Foxp3⁺Treg cells/CD4⁺ cells in synovium (23.12%±4.93% vs 24.66%±5.82%, P>0.05) was observed, whereas the ratio in TNFR-Fc treatment group was significantly increased(33.07%±5.14%). The ratio of CD4⁺RORγt⁺Th17 cells/CD4⁺ cells in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (9.74%±2.23% vs 1.00%±0.59%, 5.63%±1.76%, P<0.01). CONCLUSION: Differentiation disturbance of Treg/Th17 exists in the synovium of CIA rats. TNFR-Fc may restore the balance of Treg/Th17 by inhibiting Th17 cell differentiation and inducing the production or accumulation of Treg.     

Expression of IgG in bladder carcinoma and the effect of the antibody against human IgG on tumor cell apoptosis

AIM: To investigate the expression of immunoglobulin G (IgG) in bladder transitional carcinoma cells (BTCC) and the effect of the antibody against human IgG on tumor cell apoptosis.METHODS: The expression of IgG of BTCC was detected by immunohistochemistry, Western blotting, enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM). The expression of IgG mRNA was tested by hybridization in situ method and RT-PCR in vitro. Antiproliferation effects of the antibody of antihuman IgG on T24 and BIU-87 cell lines were measured by MTT assay. Cell apoptosis was assessed by FCM.RESULTS: The expression of IgG was 91.1％ (51/56) in clinical cases, and 45.4％ (5/11) in normal controls. Immunohistochemistry and Western blotting showed that the IgG was significantly expressed in T24 and BIU-87. FCM indicated the IgG was mainly expressed in cytoplasma. No IgG was detected by ELISA in supernatant of cell culture medium. RT-PCR and hybridization in situ demonstrated IgG mRNA was significantly expressed in two cell lines. Under the treatment of 25 mg/L of goat nonspecific IgG and the antibody of antihuman IgG, the inhibition ratio of cell growth in T24 and BIU-87 were (4.73±3.73)% vs (24.98±3.81)% and (5.36±1.53)% vs (22.70±3.72)%, respectively (P<0.05). The percentages of apoptotic cells in T24 and BIU-87 were 2.3% vs 20.7% and 1.3% vs 15.3%, respectively.CONCLUSION: IgG is significantly expressed in bladder transitional carcinoma cell. The antibody of antihuman IgG inhibits cancer cell growth and induces tumor cell apoptosis.     

Differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro via cardiotrophin 1

AIM: To investigate the effects of cardiotrophin 1 (CT-1) on differentiation of swine bone marrow mesenchymal stem cells (MSCs) into cardiomyocyte-like cells in vitro.METHODS: MSCs were isolated and proliferated from Tibet miniswine. Adipogenic and osteogenic potentials were identified. MSCs were divided into 4 groups for induction: untreated group, 5-azacytidine (5-Aza) group,CT-1 group and 5-Aza combined with CT-1 group. After induction for 4 weeks, the expression of cardiac cell markers including α-actin and cardiac troponin-T (cTnT) was estimated by immunofluorescence staining. Finally, the rates of red fluorescence positive-staining cells were calculated. RESULTS: The expression of α-actin in the 4 groups by red fluorescence staining was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 29.90%±4.76%, significantly higher than that in 5-Aza group (17.73%±2.34%, P<0.01), CT-1 group (6.63%±0.55%, P<0.01) and untreated group (1.62%±0.09%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.05). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01). The expression of cTnT in the 4 groups was as follows: the differentiation rate of cardiomyocyte-like cells in combination group was 36.50%±4.09%, significantly higher than that in 5-Aza group (14.37%±1.65%, P<0.01), CT-1 group (7.50%±0.61%, P<0.01) and untreated group (1.12%±0.23%, P<0.01). The differentiation rate in 5-Aza group was significantly higher than that in CT-1 group (P<0.01) and untreated group (P<0.01). The differentiation rate in CT-1 group was significantly higher than that in untreated group (P<0.01).CONCLUSION: Appropriate concentrations of 5-Aza (10 μmol/L) and CT-1 (0.1 μg/L) induce swine bone marrow MSCs to differentiate into cardiomyocyte-like cells in vitro. CT-1 combined with 5-Aza significantly increases the differentiation rate.     

Effect of activation of stimulator cells on expression of CD69 by responder T cells in mixed lymphocyte reaction

AIM: To study the influence of status of stimulator cells on activation of responder T cells in mixed lymphocyte reaction (MLR), so as to provide some basis for clinical transplantation. METHODS: Stimulator cells were pretreated differently before mixed lymphocyte culture (MLC) to change their functional status, fluorescence conjugated antibodies and flow cytometry were used to detect expression of CD69 by responder T cells at several different time points.RESULTS:The expression percentages of CD69 by responder T cells in MLCa group(stimulator cells were pre-activated)were significantly higher than those in MLC group(stimulator cells were not pre-activated)at 24,48and 72 hours of culture,respectively(5.21%±0.24%vs 1.98%±0.33%,29.81%±0.85%vs 20.65%±1.00%and 39.61%±1.62%vs 13.49%±0.60%,P<0.01). CONCLUSION: Our results showed that with pre-activated stimulator cells, expression of CD69 by responder T cells could be significantly elevated in MLR.     

Effect of mineralocorticoid receptor gene silencing by RNA interference on proliferation and apoptosis of murine macrophage cell line RAW 264.7

AIM: To establish stable knockdown of mineralocorticoid receptor (MR) expression through short hairpin RNA (shRNA)-mediated silencing in murine RAW 264.7 macrophages. METHODS: Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome-mediated transfection, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418-resistant cells were verified by immunofluorescent microcopy and real-time PCR, respectively. Proliferative activity of MR-silencing cell line was analyzed by CCK-8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS: MR gene expression was down-regulated by 70% compared with the negative control (NC) plasmid transfection. In addition, MR-silencing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells (P<0.05), along with reduced proliferation index of 31.0%±1.3% (P<0.05), compared with the wide type cells (37.2%±0.5%) and the NC cells (37.5%±1.6%). In resting state, the apoptotic rate in wide type, NC and MR-silencing cells were 2.18%±0.36%, 6.65%±0.81% and 7.70%±1.34%, respectively, and no statistical difference was observed between NC and MR-silencing cells (P>0.05). CONCLUSION: MR gene silencing inhibits the proliferation of RAW 264.7 macrophages, but has no obvious effect on the apoptosis of the resting state cells.     

P-selectin on activated platelets promotes hematogenous metastasis of primary lung cancer via P-selectin glycoprotein ligand-1

AIM: To investigate the relationship between platelets/P-selectin on activated platelets and clinico-pathological features, hematogenous metastasis and prognosis of lung cancer, and to explore the effect of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interaction on the hematogenous metastasis-related integrin β3 (ITGB3). METHODS: The expression of P-selectin on activated platelets was detected by flow cytometry, and its effects on lung cancer and the risk of hematogenous metastasis were analyzed. The expression of PSGL-1 in different types of lung cancer tissues and cell lines was determined by Western blot. By co-culturing platelets and lung cancer cells in vitro, the effects of up- and down-regulation of PSGL-1 on invasion and migration abilities of lung cancer cells were observed. RESULTS: The peripheral blood platelet counts and P-selectin expression on activated platelets in the patients with lung cancer were significantly increased (P<0.05). The P-selectin expression on activated platelets was significantly associated with hematogenous metastasis of lung cancer (r=0.256, P<0.05). The strongest expression of PSGL-1 was found in the lung adenocarcinoma samples, next in the lung squamous-cell carcinoma samples, and the weakest in small-cell lung cancer samples. P-selectin promoted transmembrane invasion of lung cancer cells. Inhibition of P-selectin and its ligand PSGL-1 reduced ITGB3 expression, invasion and migration of lung cancer cells. CONCLUSION: The P-selectin level on activated platelets is significantly associated with hematogenous metastasis of lung cancer, which is related to the binding of P-selectin and its ligand PSGL-1. Up-regulation of ITGB3 level after their binding might be one of the mechanisms of the remodeling of extracellular matrix to facilitate hematogenous metastasis of lung cancer.     

Rat mesenchymal stem cells differentiate into neuron-like cells with adrenaline hormones

AIM: To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%±4.3%), and differentiating cells (treated group: adrenaline 74.7%±2.6%; noradrenaline 75.9%±2.4%; isoprenaline 72.1%±4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%±6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.     

Nitric oxide opens the second window of protection in ischemic preconditioning via induction of heat shock protein 72

AIM:To examine the inhibition of nitric oxide (NO) synthesis during ischemic preconditioning (IP) on the induction of heat shock protein 72 (HSP72) and infarct size-limiting effect of the second window of protection. METHODS:Rabbits were subjected to 4 cycles of 5 min of coronary artery occlusion separated by 10 min reperfusion, or received a sham operation. During this procedure, N^G-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthase) was injected intravenously 5 min before IP followed by its continuous infusion. Twenty-four hours later, the hearts were rapidly excised for assaying HSP72 expression or were subjected to 30 min coronary artery occlusion followed by 120 min reperfusion and then measured infarct size (IS). RESULTS:Twenty-four hours later, immunoblotting revealed an increase in HSP72 protein levels in the IP group, and this was blocked by L-NAME. IS of the IP rabbits was reduced as compared with the control (29.8%±3.7% vs 50.8%±4.3%, P＜0.01). IS in the IP rabbits was elevtated as a result of L-NAME treatment (46.0%±5.1%). Administration of L-arginine reversed the effects of L-NAME on the induction of HSP72 and IS (33.5%±4.0%). The intravenous administration of S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) increased the induction of HSP72 and reduced IS (31.3%±5.7%, P＜0.01vs control) 24 h later. CONCLUSION:These findings suggest that NO may be involved in the induction of HSP72 and the opening of the second window of protection of IP.     

Elevated levels of serum IL-6, ICAM-1 and P-selectin in stable survivors with liver transplantation

AIM: To study the profile of serum IL-6， ICAM-1 and P-selectin in stable survivors with clinical liver transplantation (LTx). METHODS: Flow cytometric analysis was used to determine the phenotype of T cell subsets in peripheral blood mononuclear cells （PBMCs） from stable survivors with liver transplantation (n=22), and healthy volunteers (n=12). Serum levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF-α) and interleukin (IL-6), intercellular adhesion molecules (ICAM-1) and P-selectin in stable survivors with liver transplantation and healthy volunteers were assessed by enzyme-linked immunoabsordent assay (ELISA). Recently performed 6 cases of liver transplantation were also dynamically observed in this study. RESULTS: Percentage of CD4+ T cells， CD8+ T cells and CD3+ T cells, as well as ratio of CD4 to CD8 were no difference between two groups (P>0.05). However, a significant higher percentage of CD3+CD25+ T cells was found in stable liver transplantation group as compared to healthy group (P<0.05). Significantly increased concentrations of IL-6, ICAM-1 and P-selectin were found in stable liver transplantation group as compared to healthy group (P<0.05). A high TNF-α level was detected in stable liver transplantation group while no significant difference was found as compared to healthy volunteers group (P>0.05). There was not found no regular change of serum cytokines (IL-6, TNF-α) and adhesion molecules (ICAM-1, P-selectin) in 6 liver transplanted patients during post-operation from day 1 to day 30, indicating that was associated with the different status of patients before or after transplantation. CONCLUSIONS: Our data suggesting that increased levels of ICAM-1 and P-selectin, appears to participate in the processing of immunoregulation to transplanted livers, whereas elevated concentrations of IL-6 appear to be involved in the repair of the injury induced by TNF-α in allo-transplanted livers.     

Variation of serum NSE and S-100 β in patients with acute cerebral infarction subtypes

AIM: To explore clinical significance of the serum changes of neuron-specific enolase (NSE)and S-100 β protein (S-100 β) during acute cerebral infarction. METHODS: 59 acute cerebral infarction patients were classified as total anterior circulation infarcts (TACI), partial anterior circulation infarcts (PACI), lacunar infarcts (LACI) and posterior circulation infarcts (POCI). Their serum NSE and S-100 β concentrations were determinated by enzyme linked immunosorbent assay (ELISA) during stroke onset 6 d, and compared with 32 controls. RESULTS: The every time point serum NSE concentration of TACI was higher than controls (P＜0.01), and its highest value occured at 3 d after the onset. The every time point concentration of PACI was also higher than controls (P＜0.05), its highest value occured at 1 d after the onset. The increment of serum S-100 β synchronized with serum NSE change in TACI. The S-100 β of PACI started to increase at 3 h after the onset, its highest value occured at 1 d after the onset, and concentration at 6 h, 1 d, 3 d and 6 d was markedly higher than controls (P＜0.05). However, the every time point NSE and S-100 β concentrations of LACI and POCI increased unmarkedly compared with control group. CONCLUSIONS: The serum NSE and S-100 β changes in acute period (contains acute early period) of cerebral infarction subtypes might be different. These results might help to treat acute cerebral infarction according to the classification.     

Pathophysiologic changes of local ischemic coronary artery and cardiac muscle after ligating canine LAD

AIM: To evaluate the reliability of making a research model of coronary artery stenosis and local myocardial infarction reproduced in dog by ligating canine LAD. METHODS: We disparted 30 aged healthy cross-breed dogs ［(18.5±6.7) kg］ into three groups. The near part of the LAD through left minimal thoracic incision was ligated to interdict 25% (group A), 50% (group B), 75% (group C) of the flux, respectively. The changes of plasma endothelium-derived factors NO, ET-1, sP-selectin and CTnT were measured before ligation and at different time points after ligation. The expression of P-selectin gene in cardiac muscle was detected by Western blotting. The segments of distal parts of the ligated LAD were cut and pathological changes of the patches of topical cardiac muscle were observed by electronic microscope. RESULTS: After ligation, NO/ET-1, P-selectin and CTnT had significant changes in group B (P<0.05) and group C (P<0.01). The expression of P-selectin of cardiac muscle was highly up-regulated after ligating in B (50%) and C (75%) group, In C group animals, a typical far more intense expression pattern was found. Under electronic microscope, the endothelium and other structures of the LAD wall and ultrastructure of myocardial cells had obvious changes in later two groups, especially in group C. There were a typical stenosis of LAD and myocardial infarction. CONCLUSIONS: Ligating the LAD 75% severely damages the endothelial cell and cardiac muscle cells of local ischemic vessel and cardiac muscle, thus forms the typical local stenosis of coronary artery and myocardial infarction, such method is a safe and reasonable way for making a disease model for studying CABG in surgery.     

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.