Relationship between expression of chemokine receptors CCR3, CCR5 and CXCR3 on CD4+ T cells and spontaneous abortion in mice

Background Previous studies have shown that local immune cells in the feto-maternal interface are recruited from peripheral blood, and that chemokines and their receptors play an initial and key role in this recruitment process. In this study, we aimed to determine whether spontaneous abortion is associated with the expression of chemokine receptors CCR3, CCR5, and CXCR3 on CD4^＋ T cells.
Methods Peripheral blood, spleen, and thymus were collected from the spontaneous abortion mouse model CBA/JxDBA/2 （SA group, n=14）, the normal pregnant mouse model CBA/JxBALB/c （NP group, n=13）, and normal non-pregnant CBA/J mice （NNP group, n=11）. The number of chemokine receptors CCR3, CCR5, and CXCR3 expressed on CD4^＋ T cells was measured by double-label flow cytometry （FCM） method.
Results In peripheral blood, the SA group had significantly lower CCR3 expression （P 〈0.01） and higher CCR5 and CXCR3 expression （P 〈0.01） on CD4^＋ T cells than did the NP group. But comparing these chemokines between the SA and NNP groups, there was no significant difference （P 〉0.05）. In spleen, the SA group expressed significantly lower CCR3 expression （P 〈0.01） and higher CCR5 and CXCR3 expression （P 〈0.05） on CD4^＋ T cells than did the NP group. When compared with the NNP group, the SA group had significantly higher CCR3 expression （P 〈0.01）, but was not statistically different with regards to the other two chemokines （P 〉0.05）. In thymus, the SA group had significantly lower CCR3 expression （P 〈0.05） and higher CXCR3 expression （P 〈0.05） on CD4^＋ T cells than the NP group, with no significant difference in CCR5 expression （P 〉0.05）. Compared with the NNP group, the SA group had higher CCR3 expression （P 〈0.01）, but there was no statistical difference in CXCR3 and CCR5 expression （P 〉0.05） between the two groups.
Conclusion The abnormal expression of CCR3, CCR5 and CXCR3 on CD4^＋ T cells may play an important role in the pathogenesis of spontaneous abortion.     

Adoptive transfer of FTY720-treated immature bone marrowderived dendritic cells(BMDCs) significantly reduced the spontaneous resorption rate in the CBA/J ×DBA/2 mouse model

Objective To investigate the effect of FTY720-treated immature bone marrow-derived dendritic cells(BMDCs) on the embryo resorption rate in the CBA/J× DBA/2 abortion mouse model.Methods The dendritic cells(DCs) were derived from bone marrow of DBA/2 mice, and then co-cultured with FTY720. The abortion mouse models were established by mating female CBA/J mice with DBA/2 mice. Via the CBA/J×DBA/2 abortion mouse model, six groups were established, group A: normal pregnancy model; group B: abortion mouse model with no treatment; group C: abortion mouse model injected with DC culture medium(DCCM); group D: abortion mouse model injected with DC; group E: abortion mouse model injected with FTY720; group F: abortion model mouse injected with FTY720-DC. The differences were compared in the embryo resorption rates of the CBA/J ×DBA/2 abortion mouse model treated with FTY720-DC or different controls observed on gestation day 12 to 14, and then the microenvironment in murine pregnancy was investigated.Results The embryo resorption rate was statistically significantly decreased in group D and group E when they compared with group B and group C(P0.05, respectively).Furthermore, the embryo resorption rate in group F showed a statistically significant decrease when compared with the other groups except group A(P0.01). These resultssuggest that FTY720-DCs possess a notable advantage over DCs or FTY720 in reducing the embryo resorption rate of the abortion mouse model. The percentage of Th17(IL-17+CD4+T cells) in peripheral blood mononuclear cell(PBMC) in the abortion mouse model was 4.35%±0.34% before treated with FTY720-DC, and was1.34%±0.28% after treated with FTY720-DC(P0.01). The percentage of Tregs(CD4~+CD25~+Foxp3~+T cells) in PBMC was significantly increased in group F(8.35%±1.80%) as compared with group B(2.68%±0.65%)(P0.01).Conclusion Adoptive transfer of FTY720-DC can statistically significantly reduce the embryo resorption rate in the CBA/J×DBA/2 abortion mouse model. The lower embryo resorption rate in the FTY720-DC treated abortion mouse model may be caused by the imbalance of Treg/Th17.     

Effect of TGF-β1 on the Expression of IL-12, IL-15, IL-18, IL-4 and IL-10 in Heart Transplantation Rejection in Rats

To investigate the effect of TGF-β1 on the expressions of IL-12, IL-15, IL-18, IL-4 and IL-10 in heart transplantation rejection in rats, a model of rat cervical heterotopic heart transplantation was set up and the model rats were randomly divided into three groups： control group, transplant group and TGF-β1 group. The mRNA expression levels of IL-12, IL-15, IL-18, IL-4 and IL-10 were determined by RT-PCR at the 5th day after the transplantation. The mRNA expression levels of IL- 12, IL-15, IL-18 were increased obviously and those of IL-4, IL-10 were significantly decreased in the transplant group as compared with the control group （P〈0.01）. In the TGF-β1 group, the mRNA ex- pression levels of IL- 12, IL- 15, IL- 18 were significantly decreased and those of IL-4, IL- 10 were significantly increased as compared with the transplant group （P〈0.01）. The immunosuppressive effect of TGF-β1 on heart transplantation rejection was related to its inhibition of the expressions of Th1-type cytokines （IL-12, IL-15, IL-18 etc） and its promotion of the expressions of Th2-tpye cyto- kines （IL-4, IL-10）.     

The Relationship between Murine Spontaneous Miscarriage and Lymphocyte Infiltration at the Fetomaternal Interface

Objective To assess the relationship between murine recurrent spontaneous abortion and lymphocyte infiltration at the fetomaternal interface Materials & Methods Hysterolaparotomy was performed on d13. 5 of murine gestation, and resorption rate of embryos was calculated in two different mating combinations of abortion model CBA/J × DBA/2 and of the fertile control CBA/J × BALB/c,respectively. CD69 was used as an activation marker on NK cell surface, while DX5 was used as a common marker of NK cell in this study. The proportions of CD 69+ cells in isolated lymphocytes, and CD 69+ DX 5+ cells in DX5+ subpopulation at the fetomaternal interface were determined by two-color flow cytometry. It was further evaluated whether there was linear correlation between these cell proportions and resorption rate of embryos.Results Resorption rate of embryos were 36. 0% and 7. 2% (P ＜ 0. 01) in CBA/J ×DBA/2 and CBA/J × BALB/c (genotypes are all of H-2k × H-2d) , respectively. In CBA/J × DBA/2 mice, no linear correlation was observed between the proportion of CD69+ cells in lymphocytes (x) and resorption rate (y) (r= 0. 4054). However, it appeared that the proportion of CD69+DX 5+ cells/DX5+ cells (x) was strongly linear correlated with the resorption rate of embryos (y) (r = 0. 8156, y = - 0. 3958+2. 1237x) in this model.Conclusion Infiltration of CD 69+ DX5+ cells at the fetomaternal interface may be associated with recurrent embryo-resorption in CBA/J × DBA/2 mice.     

Relationship between the expression of Toll-like receptor 2 and 4 in mononuclear cells and postoperative acute lung injury in orthotopic liver transplantation

Background The aim of this study was to investigate the potential relationship between the dynamic expression of Toll-like receptor 2 and 4 （TLR2/4） in peripheral blood mononuclear cells as well as changes in serum concentration of inflammatory factors and acute lung injury （ALl） in patients after orthotopic liver transplantation （OLT）. Methods The peripheral blood samples of 27 patients （23 men and 4 women with ASA Ⅲ to Ⅳ） who received OLT were collected for measurement of TLR2/4 at T1 （after induction of anesthesia）, T2 （25 minutes after anhepatic phase）, T3 （3 hours after graft reperfusion） and T4 （24 hours after graft reperfusion）. The expression of TLR2/4 in mononuclear cells was measured by flow cytometry. The serum concentrations of tumor necrosis factor （TNF）-α, interleukin （IL）-1β and IL-8 were measured by enzyme-linked immunosorbent assay （ELISA）. Twenty-seven patients were assigned to ALl group （n=-9） and non-ALl group （n=-18） according to the diagnostic criteria of ALl. The expression of TLR2/4 in the ALl group or non-ALl group was analyzed. Results Compared to the non-ALl group, the volumes of blood loss, ascites, total output and transfused red blood cells were higher in the ALl group, and the anhepatic phase lasted longer （P 〈0.05, P 〈0.01）. The expression of TLR2/4 in mononuclear cells increased significantly at T3 and T4, and serum concentrations of TNF-α, IL-1β and IL-8 increased significantly too. There was no significant difference in Child-Turcotte-Pugh （CTP） scores between the ALl group and non-ALl group （P 〉0.05）. The expression of TLR2/4 in mononuclear cells increased significantly at T3 and T4 in the ALl group （P 〈0.05, P〈0.01）. A positive correlation was noted between the expression of TLR4 in mononuclear cells and the serum concentrations of TNF-α, IL-1β （P=0.041, P=0.046） in the ALl group. In the non-ALl group, statistical results showed that the expression level of TLR2/4 in mononuclear cells was not significantly different during the peri-operative period of OLT （besides TLR4 expression at T4）. Compared to expression in mononuclear cells was more significant in the mononuclear cells exceeded that at T1 by one time were more 16. the non-ALl group, the increasing amplitude of TLR2/4 ALl group. The patients whose TLR2/4 expression in likely to suffer from ALl （P=0.013）, with a relative risk of Conclusion The expression level of TLR2/4 in mononuclear cells increases significantly in the peri-operative period of OLT, and it may be a high risk factor for occurrence of postoperative ALl.     

Inhibitory Effects of Parthenolide on the Angiogenesis Induced by Human Multiple Myeloma Cells and the Mechanism

The inhibitory effects of parthenolide （PTL） on angiogenesis induced by multiple myeloma （MM） cells in vitro and the mechanism were investigated. Human MM line RPMI8226 cells were cultured in vitro. The effects of MM culture supernatant on the migration and tubule formation ability of human umbilical vein endothelial cells （HUVECs） treated with PTL were observed. By using Western blot, the expression of p65 and IкB-α in MM cells was detected. RT-PCR was used to assay the expression of VEGF, IL-6, MMP2 and MMP9 mRNA in MM cells. ELISA was used to measure the levels of VEGF and IL-6 in MM cell culture supernatant. The expression of MMP2 and MMP9 in MM cells was examined by immunohistochemistry. （1） In 3.5, 5.0, 7.5 and 10 μmol/L PTL groups the number of migrated cells was 310±56, 207±28, 127±21 and 49±10 respectively, which was significantly different from that in positive control group （598±47） （P〈0.01）. In 3.5 and 5.0 μmol/L PTL groups the areas of capillary-like structures were 0.092±0.003 and 0.063±0.002 mm2, significantly less than in positive control group （0.262±0.012 mm2） （P〈0.01）, but in 7.5 and 10 μmol/L PTL groups no capillary-like structures were found; （2） After treatment with different concentrations of PTL for 48 h, the expression of p65 protein was gradually decreased, while that of IкB-α was gradually enhanced with the increased concentration of PTL; （3） After treatment with 3.5, 5.0, 7.5 and 10 μmol/L PTL for 48 h, the VEGF levels in the supernatant were 2373.4±392.2, 1982.3±293.3, 1247.0±338.4 and 936.5±168.5 pg/mL respectively, significantly different from those in positive control group （2729±440.0 pg/mL） （P〈0.05）. After treatment with 7.5 and 10 μmol/L PTL, the IL-6 levels in the culture supernatant were 59.6±2.8 and 41.4±9.8 pg/mL respectively, signifi- cantly lower than in positive control group （1287.3±43.5 pg/mL） （P〈0.05）; （4） RT-PCR revealed that PTL could significantly inhibit the expression of V     

Effect of Wumeiwan on Cytokines TNF-α, IL-6, IL-8, IL-10 and Expression of NF-κBp65 in Rats with Ulcerative Colitis

The effects of Wumeiwan （WMW） on TNF-α, IL-6, IL-8, IL-10 and NF-κBp65 in rats with ulcerative colitis （UC） were investigated, the curative effectiveness of WMW vs salicylazosulfapyridine （SASP） was compared, and the action mechanism was analyzed. Fifty-Six Sprague-Dawley （SD） rats were randomly divided into four groups （n=14 in each group, with equal ratio of male and female）： normal control group, model group, SASP group, and WMW group. Except normal control group, the rat UC models in the remaining three groups were established using the method of 2.4-dinitrochlorobenzene （DNCB） immunization and acetic acid local enema. The rats in model group, SASP group, and WMW group were treated with distilled water, SASP, and WMW respectively. The changes in the symptoms and signs were observed, and levels of IL-6, IL-8, TNF-α, IL-10 and the expression of NF-κBp65 in the colonic tissues were statistically analyzed. The results showed that the levels of IL-6, IL-8, and TNF-α were significantly increased （P〈0.01）, while those of IL-10 significantly reduced （P〈0.01） after establishment of rat UC models as compared with normal control group. The levels of IL-6, IL-8, and TNF-α were obviously lower, but the level of IL-10 was obviously higher in WMW and SASP groups than those in model group （P〈0.05）. The levels of IL-6, IL-8, and TNF-α were lower, while the level oflL-10 was higher in WMW group than in SASP group. NF-κBp65 was expressed negatively or weakly in normal colonic tissues. The positive expression rate of NF-κBp65 in WMW group and SASP group was obviously lower than in model group （P〈0.01）, and there was significant difference between WMW group and SASP group （P〈0.05）. It was concluded that rat UC model was established successfully. WMW could up-regulate the expression of IL-10, down-regulate the expression of TNF-α, IL-6, IL-8, and inhibit the NF-κBp65 activity to adjust immune function, indicating WMW had better curative effects on UC in rats.     

Small interfering RNA-mediated knockdown of Notch1 in lung

Background The immunologic response to allergens mediated by T lymphocytes is an incipient key element in the pathogenesis of asthma, and Thl/Th2 balance is regarded as the core of asthma pathogenesis. Notch is a single-pass transmembrane receptor protein that regulates differentiation, proliferation and apoptosis in a broad range of cells. It is considered that the Notch signal pathway works in every stage of T cell development and differentiation. Whether the pathway of asthma pathogenesis is related to Notch1 remains unknown. This study is aimed to investigate whether the pathway of asthma pathogenesis is related to Notch1 by examining the effect of knockdown of the Notch1 gene by small interfering RNA on T cell differentiation. Methods An OVA-induced asthma mouse model was established. The expression of Notch1 in the tissue and T cells of the lung from asthmatic mice was detected by RT-PCR and Western blotting. The expression of Notch1 and cytokine interleukin （IL）-4 and interferon （IFN）-γ in activated lung T cells was detected by RT-PCR and enzyme-linked immunosorbent assay after blocking Notch1 by small interfering RNA. Results The mRNA and protein expression of Notch1 increased significantly both in the lung tissue and lung T cells of asthmatic mice （both P 〈0.05）. IL-4 decreased and IFN-y increased significantly in active lung T cells after Notch1 was blocked by Notchl-specific small interfering RNA （IL-4： （2.51±0.51） pg/ml vs 0.64±0.27） pg/ml protein; IFN-γ： （21.72±4.24） pg/ml vs （39.79±4.09） pg/ml protein, P 〈0.05）. Conclusion This study demonstrated that the Notch1 signal might play a role in the pathogenesis of asthma by its involvement in Thl/Th2 differentiation.     

Activation of NF-κB and its regulation on IL-6 expression during LPS-induced liver injury

Objective： To explore the kinetics of the activation of nuclear factor-kappa B （NF-κB） and its regulation of interleukin-6 （IL-6） expression during LPS induced liver injury. Methods： Kunming mice were randomly divided into 4 groups in order to observe the does effect relationship at 3h： normal saline solution （control） group, low （1 mg/kg）, middle （5 mg/kg）, and high （10 mg/kg） LPS-induced groups; 6 groups in order to observe the time-effect relationship of 5 mg/kg LPS injection： normal saline solution （control） group, 0.5, 1, 3, 5 and 8 h groups ; pyrrolidine dithiocarbamate （PDTC） intervened groups （3 h）： normal saline solution （control） group, 5 mg/kg LPS, 200 mg/kg PDTC, and 200 mg/kg PDTC＋5 mg/kg LPS groups. NF-κB activities of Kupffer cells were determined with electrophoretic mobility shift assay （EMSA） and expression levels of IL-6 were measured with enzyme-linked immunosorbent assay （ELISA）. Results： Does-effect of NF-κB activities in Kupffer cells after LPS injection 3 h： NF-κB activation could be detected in 1 mg/kg LPS group, reached the highest level in 5 mg/kg LPS group, and persisted in 10 mg/kg LPS group; time-course after 5 mg/kg LPS injection： the DNA-binding activity was observable at 0.5 h after LPS injection, increased significantly at 3 h, and persisted for at least 8 h; in addition, antioxidant PDTC could inhibit the activation of NF-κB significantly. The kinetics of IL-6 level in liver tissues during LPS-induced liver injury were that IL-6 level after 3 h of injection increased first and then reduced; the same trend was observed in the time-course on IL-6 level after LPS injection; PDTC could significantly inhibit the release of IL-6. Correlation analyses revealed that IL-6 level was significantly and positively correlated with the activation of NF-κB. Conclusion ： NF-κB in Kupffer cells can be activitied during LPS-induced liver injury to some extent, and NF-κB may have some regulation on the expression of IL-6.     

Effects of propofol on the mRNA expression of toll-like receptor 4 in BV-2 cells during mimic ischemia-reperfusion injury <Emphasis Type="Italic">in vitro</Emphasis>

This study investigated the effects of propofol on the mRNA expression of Toll-like receptor-4 （TLR4） in BV-2 cells during mimic ischemia-reperfusion （I/R） injury in vitro. BV-2 cells, a mouse microglia line, were cultured and divided into 4 groups at random： control group （group C）, ischemia/reperfusion group （group I/R）, low-dose propofol （25 μmol/L） intervention group （group PF25） and high-dose propofol （100 μmol/L） intervention group （group PF100）. The mRNA expression of TLR4 and NF-κB was measured by means of RT-PCR. TNF-α levels in the supernatants of BV-2 cells were detected by ELISA. The results showed that the mRNA expression of TLR4 and NF-κB was significantly higher in groups I/R, PF25 and PF100 than in group C （P〈0.01）. And the TNF-α level in the supernatants was elevated in groups I/R, PF25 and PF100 as compared with that in group C （P〈0.01）. After pre-treatment with propofol, the mRNA expressions of TLR4 and NF-κB and the TNF-α level were significantly decreased in groups PF25 and PF100 in comparison to those in group I/R （P〈0.01）. And the decrease in those indicators was more significant in group PF100 than in group PF25 （P〈0.01）. It was concluded that propofol exerted brain-protecting effects during I/R injury by suppressing the mRNA expressions of TLR4 and NF-κB and deceasing the TNF-α level.     

Changes in Number and Biological Function of Endothelial Progenitor Cells in Hypertension Disorder Complicating Pregnancy

To examine the changes in number and function of endothelial progenitor cells （EPCs） from peripheral blood （PB） in hypertension disorder complicating pregnancy （HDCP）, 20 women with HDCP and 20 normal pregnant women at the third trimester were studied. Mononuclear cells （MNCs） from PB were isolated by Ficoll density gradient centrifugation. EPCs were identified by positive expression of both CD34 and CD133 under fluorescence microscope and positive expression of factor Ⅷ as shown by immunocytochemistry. The number of EPCs was flow-cytometrically determined. Proliferation and migration of EPCs were measured by MTT assay and modified Boyden chamber assay, respectively. The adhesion activity of EPCs was detected by counting the number of the adherent cells. The results showed that, compared with normal pregnant women, the number of EPCs was significantly reduced in HDCP （4.29%±1.21% vs 15.32%±2.00%, P〈0.01）, the functional activity of EPCs in HDCP, such as proliferation （13.45%±1.68% vs 18.45%±1.67%）, migration （37.25±7.28 cells/field vs 67.10±9.55 cells/field） and adhesion activity （20.65±5.19 cells/field vs 34.40±6.72 cells/filed） was impaired （P〈0.01）. It is concluded that the number and function of EPCs are significantly decreased in HDCP.     

Changes of Regulatory T Cells in Graves＇ Disease

The immune mechanism of Graves＇ diseases （GD） and the roles of regulator T cells were investigated. In 32 patients with GD （GD group） and 20 healthy volunteers （control group）, flow cytometry was used to detect the proportion of CD4^＋CD25^＋ cells, MACS to isolate CD4^＋ CD25^＋ cells, RT-PCR to assay the expression of FOXP3, and ELISA to test the leyel of IL-10, respectively. It was found that there was no significant change in the proportion of CD4^＋CD25^＋ T cells between GD group and control group （P〉0.05）, while secretion of IL-10 and expression of FOXP3 in GD group were lower than control group （P〈0.01 and P〈0.05, respectively）. In conclusion, though the proportion of regulatory T cells of peripheral blood lymphocytes in the patients with GD, the functions of them were significantly weakened, which might be a pathogenic factor in GD.     

The Effect of Targeted Magnetic Nanopaticles on Hepatoma and the Expression of bcl-2/bax Protein

The effect of targeted magnetic nanoparticles on hepatoma and the underlying mechanism were examined. Nude mice transplanted with a human hepatoma cell line （HepG2 cells） were randomized into 5 groups, including： （1） group A, receiving normal saline, （2） group B, receiving 5-fluorouracil （5-Fu）, （3） group C, receiving magnetic nanoparticles containing 5-Fu, （4） group D, consisting of treatment with magnetic nanoparticles containing 5-Fu and inside magnetic field and （5） group E, receiving pure magnetic nanoparticles and inside magnetic field. Morphological features of transplanted tumors in mice in each group were observed under transmission electron microscope （TEM）. The expression of bcl-2/bax protein was immunohistochemically detected by SABC method. The results showed that a large number of apoptotic tumor cells were found in group B and group D under TEM. The expression of bcl-2 protein was significantly decreased and the expression of bax protein increased significantly in both group B and D as compared with those in group A, C and E （P〈0.01 for all）. The decrease in bcl-2 and the increase in bax were more in group D as compared with group B （P〈0.01）. It is concluded that the targeted magnetic nanoparticles containing 5-Fu can improve the chemotherapeutic effect of 5-Fu by decreasing bcl-2 expression, increasing bax expres- sion and inducing apoptosis of the liver cancer cells.     

The Effect of Ginkgo Biloba Extract on the Expression of PKCα in the Inflammatory Cells and the Level of IL-5 in Induced Sputum of Asthmatic Patients

To investigate the effect of the Ginkgo Biloba Extract （GBE） on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticasone propionate for 2 weeks or 4 weeks, or treated with fluticasone propionate plus GBE for 2 weeks or 4 weeks. Fifteen healthy volunteers served as healthy controls. Sputum inhalation with inhaling hypertonic saline （4%-5%） was performed. Lung ventilatory function and forced expiratory volume in one second （FEVI） were measured. The numbers of different cells in induced sputum were calculated. The expression of PKCα in the cells was immunocytochemically detected and the percentages of positive cells in different cells were counted. Interleukin-5 （IL-5） in sputum supernatants was detected with enzyme-linked immunosorbent assay. The percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the concentration of IL-5 in asthmatic patients were higher than those in the controls （P〈0.05）, and the eosinophils, lymphocytes, positive expression of PKCα and the level of IL-5 were significantly decreased in asthmatic patients after they were treated with fluticasone propionate or fluticasone propionate plus GBE. However, they were still significantly higher than those of the controls. Compared to the group treated with glucocorticosteroid for 2 weeks, no significant decrease was found in the percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the IL-5 in the supernatant of induced sputum. Compared with the group treated with glucocorticosteroid for 2 or 4 weeks, significant decrease in the same parameters was observed in the group treated with fluticasone propionate and GBE for 4 weeks. The IL-5 level in the supernatant of induced sputum was positively correlated with the percentage of PKCα-positive inflammatory cells and the percentage of eosinophils in the induced sputum in asthma patient groups respectively （n=150, r=0.83, P〈0.01; n=150,