Objective To establish a closed-chest pulmonary embolism-reperfusion animal model by Swan-Ganz catheter and to explore the mechanisms of pulmonary embolism(PE)-reperfusion injury(RI). Methods Experiments were made on 14 mongrel dogs,ranging in weight from 15 to 18 kg,anesthetized with 3% pentobarbital sodium.The dogs were intubated with I.D.7 endotracheal tubes.Under sterile conditions,a 7 F Swan-Ganz catheter via the external jugular vein was positioned in the unilateral pulmonary diaphragmatic lobe(DL)artery.Occlusion/reperfusion of the DL artery was controlled with 1.2 ml diluted contrast agent filled into/drawn from the balloon.After the 24 h PE,the balloon was deflated to result in 4 h reperfusion of the DL.Measurements of blood gases and tumor necrosis factor-?(TNF-?)were made at normal condition,at 24 h PE and at 4 h reperfusion.Thin-section CT scans were performed at normal condition,24 h PE,30 min,1,2,3 and 4 h reperfusion,respectively.At the end of each experiment, tissue specimens of bilateral diaphragmatic lobes were obtained for both wet/dry(W/D)weight ratio and for pathological study.Results Reperfusion pulmonary edema(RPE)was an acute,mixed,noncardiogenic edema that was observed in all 14 dogs who had been successfully established as PE/RI animal models.RPE demonstrated heterogeneous ground-glass opacifications that predominated in the areas distal to the recanalized vessels.It manifested pathologically as an edematous lung infiltrated by inflammatory cells.The mean ofPaO_2 and TNF-? of 4 h reperfusion was(81?4)mm Hg(l mm Hg=0.133 kPa)and(16.0? 2.5)pg/ml,which were significantly different(P

Cold Temperature ; Dinoprost ; analogs & derivatives ; urine ; Dust ; Humans ; Interleukin-6 ; urine ; Thromboxane B2 ; analogs & derivatives ; urine ; Weather

The establishment of high specificity and sensitivity method of small molecule monoclonal antibody-based immunoassay has a great importance in the study of small molecule compounds in Chinese medicine, wherein synthesis of small molecule artificial antigen is a critical step in the preparation of small molecule antibodies. Oxidation method using sodium iodide was used to synthesize immunogenic antigen (FRn-BSA) and coating antigen (FRn-OVA) of forsythin. UV spectroscopy and thin layer chromatography showed that forsythin was successfully conjugated with BSA and OVA. After immuned FRn-BSA, the mice could specifically produce anti-forsythin antibodies with titer up to 1:8 000, and the linear range was from 1 mg x L(-1) to 100 mg x L(-1). In this paper, the artificial antigen of forsythin was successfully synthesized, which can be applied for preparation of monoclonal antibodies and establishment of appropriate immune method.
Animals ; Antibodies ; immunology ; Antigens ; chemistry ; immunology ; Bridged Bicyclo Compounds, Heterocyclic ; chemistry ; immunology ; Drugs, Chinese Herbal ; chemistry ; Furans ; chemistry ; immunology ; Male ; Mice ; Mice, Inbred BALB C

To confirm the mechanism of exosomes as tumor vaccines inducing immunity response, dendritic cells (DCs) were induced from human peripheral blood mononuclear cells, while exosomes were isolated from DC loaded tumor antigen. The effect of exosomes on priming T cell proliferation was analysed under conditions with or without DCs, or DCs at different mature stages. The function of exosomes in immunity was detected through block test after blocking some molecules (CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83). The effect of DCs on embedded exosomes was observed by confocal microscopy, the effect of blocking surface molecules on exosomes on DC-embedding exosomes was assayed by flow cytometry. The results indicated that both exosomes derived from imDC (imDex) and exosomes derived from mDC (mDex) could not prime T cells without DC or with imDC. The exosomes derived from mDC induced with different cytokines (LPS, TNF-alpha, CpG, CD40L) were no significant difference in concentrations but were different in effect. The immunity function of exosomes depended on CD11a, CD11b, CD11c, CD54, MFG-E8 and CD83 molecules, the effect of priming T cells is reduced when these molecules were blocked. Confocal microscopy and FACS assay showed that blocking CD11a and CD54 could inhibit exosome-targeted DC and DC-embedded exosomes. It is concluded that the exosomes target DCs through their surface molecules, therefore results in immune response of T cells.
Antigens, Neoplasm ; immunology ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; secretion ; Exosomes ; immunology ; Humans ; K562 Cells ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; cytology ; immunology

The aim of this study was to examine the priming effect of sphingosine 1-phosphate (S1P) on fMLP-activated neutrophils, mainly to detect the neutrophil respiratory burst products, and to investigate the signaling pathway involved in S1P activity. Flow cytometry was used to evaluate the new isolated neutrophil; the superoxide anion output was detected indirectly by cytochrome C reduction in respiratory burst; the dihydro-rhodamine 123 was used to detect the intensity of respiratory burst; the signal transduction pathways of neutrophil respiratory burst were explored by Western blot. The results showed that after pretreated with S1P, the level of superoxide anion released by fMLP-activated neutrophils significantly increased; the Rhodamine 123 mean fluorescence intensity in S1P primed fMLP-activated neutrophils group was significantly higher than that in fMLP treatment group; PI3K and Akt proteins involved in the signal pathway of neutrophil respiratory burst. It is concluded that S1P is a new priming reagent, which primes respiratory burst of fMLP-activated neutrophils; this signal pathway may be that S1P interacts with its receptor, activates PI3K, then activates Akt-transmitting signals through NADPH oxidase, finally results in the respiratory burst.
Cells, Cultured ; Humans ; Lysophospholipids ; metabolism ; NADPH Oxidases ; metabolism ; Neutrophils ; metabolism ; physiology ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptors, Lysosphingolipid ; metabolism ; Respiratory Burst ; Signal Transduction ; Sphingosine ; analogs & derivatives ; metabolism ; Superoxides ; metabolism

OBJECTIVETo explore the role of estrogen in the regulation of the expression of claudin-6 and biological behavior in MCF-7 cells.

METHODSRT-PCR and immunocytochemistry were conducted to analyze the expression and localization of claudin-6 in MCF-7 cells treated with 17β-estradiol. CCK-8 kit assay and Scratch Test were conducted to analyze the capability of proliferation and migration of 17β-estradiol treated MCF-7 cells.

RESULTSRT-PCR analysis and immunocytochemistry showed that 17β-estradiol induced a concentration-and time-dependent effect on claudin-6. At 5 nmol/L and at 24 h, 17β-estradiol treatment led to an increased level of claudin-6, which was located in the membrane of MCF-7 cells. CCK-8 analysis showed a significant decrease in the capability of proliferation of MCF-7 cells compared with the control group (P < 0.05). Cells Scratch Test showed decreased migration capability of MCF-7 cells compared with the control group (P > 0.05).

CONCLUSIONS17β-E2 might regulate the expression of claudin-6 and inhibit the proliferation and migration of MCF-7 cells. The inhibitory effects of 17β-E2 on growth and migration of MCF-7 cells may be mediated by claudin-6 expression regulation.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Claudins ; Dose-Response Relationship, Drug ; Estradiol ; administration & dosage ; pharmacology ; Female ; Humans ; Membrane Proteins ; metabolism

OBJECTIVETo study the influence of human plasma exosomes-like vesicles on the regulatory function of macrophages.

METHODSThe exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugation and ultrafiltration. Macrophages were derived from cultured human blood monocytes. The molecular markers of macrophages were assayed by FACS. After cultured with exosomes-like vesicles, the changes of macrophages cytoplasma Ca(2+), and related genes and proteins were assayed by FACS, RT-PCR and Western Blot, respectively.

RESULTSAfter cultured with exosomes-like vesicles, mean fluorescent intensity (MFI) of macrophages cytoplasma Ca(2+) was increased. The vesicles enhanced macrophages to express cytokines genes, the expression of IL-1β and TNF-α genes being increased by 0.85 and 1.69 times respectively at 2 h, and that of IL-6 gene 3.7 times compared with the control at 8 h. However, the vesicles inhibited the expression of macrophages IL-10 gene, had no influence on the Frizzled5 receptor expression and could induce CaMKII phosphorylation.

CONCLUSIONSExosomes-like vesicles can up-regulat macrophages expression of inflammatory cytokines genes, and increase the secretion of inflammatory cytokines by activating the Wnt5A-Ca(2+) signaling pathway.

Adolescent ; Adult ; Calcium ; metabolism ; Calcium Signaling ; Exosomes ; Female ; Humans ; Macrophage Activation ; Macrophages ; metabolism ; Middle Aged ; Proto-Oncogene Proteins ; metabolism ; Wnt Proteins ; metabolism ; Wnt-5a Protein ; Young Adult

Acquired Immunodeficiency Syndrome ; prevention & control ; Adult ; China ; Drug Users ; psychology ; Female ; Health Knowledge, Attitudes, Practice ; Humans ; Male ; Needs Assessment ; Preventive Health Services ; utilization ; Risk-Taking ; Sentinel Surveillance

OBJECTIVETo identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.

METHODSThe exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration. Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry. CD4(+)T cells and CD4(+)CD25(+)CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) by Magnetic cell sorting. After exosomes-like vesicles cultured with CD4(+)T cells or CD4(+)CD25(+)CD127low Treg cells, cell proliferation and apoptosis were assayed. Phosphorylated β-catenin level in Wnt signaling by phosflow.

RESULTSExosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-II molecule, costimulatory molecules CD86 etc. After co-cultured with CD4(+) T cells, exosomes-like vesicles inhibited the proliferation of the T cells in a dose-dependent manner. After Treg cells cultured with exosomes-like vesicles for 14 days, the survival rate of the Treg cells was 57.07%, while that of the control Treg was 30.91%. Frizzled receptors 2, 3, 4and LRP6 gene mRNA expressed (the relative gray value was 48.50, 34.84, 23.85, 49.73) in the Treg cells by RT-PCR, and Wnt molecular expressed in exosomes-like vesicles. After Treg cells co-cultured with exosomes-like vesicles, the MFI of phosphorylated β-catenin decreased (from 20.06 ± 2.99 to 12.41 ± 2.08), and the expression of Bcl-2 mRNA was upregulated significantly (the relative gray value from 0.45 to 84.97).

CONCLUSIONSExosomes-like vesicles existed in human plasma and express immune regulatory molecules. They can suppress the proliferation of activated CD4(+) T cells induce their apoptosis and pro-long the survival of natural Treg cells via Wnt signaling pathway.

CD4-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Exosomes ; Flow Cytometry ; Humans ; Immunologic Factors ; Interleukin-2 Receptor alpha Subunit ; Leukocytes, Mononuclear ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo explore the biological characteristic of third-party-derived tolerogenic DC(tDC) and the influence of third-party-derived tDC on acute graft-versus-host-disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) in mice.

METHODStDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-β1D1. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. Mixed lymphocyte reaction was applied to analyze the influence of third-party-derived tDC on allo-CD4(+)T cells proliferation in vitro. Different doses of D1-tDC were adoptive transferred in the aGVHD model in allogeneic BMT which B6 mice as donors and D2 mice as recipients. Survival time, clinical GVHD score and the levels of Th1/2 cytokines in serum were monitored after allo-BMT using the aGVHD model as control.

RESULTStDC expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86 and CD40, even when stimulated by LPS. The results by RT-PCR indicated that tDC expressed low levels of IL-12p40 and high levels of immunosuppressive molecules, such as IL-10, TGF-β, Fas Ligand, indoleamine 2, 3-dioxygenase (IDO) and arginase. In the allogeneic MLR, third-party tDC suppressed allo-CD4(+)T cells proliferation, which was relative to the dose of tDC. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Remarkably, if 10(4) third-party tDC were transferred, 60% mice survived at least 60 days. When the doses of tDC were reduced to 10(3) cells, only 20% of mice survived day 60, and when increased tDC to 10(5), all of the mice died within day 37 after allo-BMT. The cytokine levels in serum indicated that 10(4) tDC-treated mice secreted in vivo high level of IL-10 21d after BMT (P < 0.05), the levels of IL-10 in 10(3), 10(4) and 10(5) tDC-treated mice were (114.23 ± 7.78), (646.18 ± 212.02), (121.97 ± 10.47) ng/L, respectively.

CONCLUSIONThird-party tDC could suppress allo-CD4(+)T cells proliferation in vitro and prevent aGVHD in allogeneic BMT mode, which may be mediated by modulating tolerogenic cytokines secretion, such as IL-10. And this effect was associated with the dose of tDC. Adoptive therapy by transfusing third-party tDC cultured with low doses of GM-CSF, IL-10 and TGF-β1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.

Animals ; Bone Marrow Transplantation ; adverse effects ; CD4-Positive T-Lymphocytes ; cytology ; Cell Proliferation ; Dendritic Cells ; cytology ; immunology ; metabolism ; Graft vs Host Disease ; prevention & control ; Interleukin-10 ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Transforming Growth Factor beta1 ; immunology ; Transplantation, Homologous