Recent studies have showed that thrombosis is closely related to leucocytes involved in immunity. Interfering with the binding of leukocyte integrin Mac-1 and platelet GPIbα can inhibit thrombosis without affecting physiological coagulation. Mac-1-GPIbα is proposed as a potential safety target for antithrombotic agents. Guanxinning tablet (GXNT) is an oral Chinese patent medicine used for the treatment of angina pectoris, which contains phenolic acid active ingredients, such as salvianolic acids, ferulic acid, chlorogenic acid, caffeic acid, rosmarinic acid, tanshinol, and protocatechualdehyde. Our previous studies demonstrated that GXN exhibited significant antithrombotic effects, and clinical studies suggested that it did not increase bleeding risk. In addition, GXN exerted a significantly regulatory effect on immune inflammation. In the current study, we intended to evaluate the effects of GXN on bleeding events and explore the safety antithrombotic mechanism of GXN based on leukocyte-platelet interaction. First, we established a gastric ulcer model induced by acetic acid in rats and found that GXN not only did not increase the degree of gastrointestinal bleeding when gastric ulcer occurred, but also had a certain promoting effect on the healing of gastric ulcer. Second, in vitroexperiments showed that after pretreatment with GXN and activation by phorbol 12-myristate-13-acetate (PMA), the adhesion and aggregation of leukocytes with human platelets were reduced. It was also found that GXN reduced the expression and activation of Mac-1 in leucocytes, and inhibited platelet activation due to leukocyte engagement via Mac-1. Overall, the results suggest that GXN may be a safe antithrombotic agent, and its low bleeding risk mechanism is probably related to inhibited leukocyte-platelet aggregation and its interaction target Mac-1-GPIbα.
Animals ; Fibrinolytic Agents ; Humans ; Integrins ; Leukocytes ; Macrophage-1 Antigen ; Rats ; Stomach Ulcer ; Tablets ; Thrombosis

OBJECTIVETo construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.

METHODSHuman IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1, and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000. The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA, and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay. The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation, and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.

RESULTSEnzyme digestion, PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB. IL-12, GM-CSF and B7.1 expressions were all detected in transfected 293T cells, and the expression of IL-12 was also detected in the transfected mouse muscular tissues.

CONCLUSIONA novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro, which facilitates further studies of tumor immunogene therapy.

Animals ; B7-1 Antigen ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; Electroporation ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Humans ; Interleukin-12 ; genetics ; immunology ; Mice ; Plasmids ; Transfection

OBJECTIVETo investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.

METHODSMice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.

RESULTSB220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].

CONCLUSIONAfter GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Interferon-gamma ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; Stomach Neoplasms ; immunology ; metabolism ; pathology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

PURPOSE: Dendritic cells (DCs) are considered the most professional antigen-presenting cells (APCs) because of their unique role in initiating immunity against threatening antigens. Recently, hypertonicity has been suggested to be involved in the activation and development of immune cells such as T cells. And tonicity enhancer binding protein (TonEBP) has been thought to play a pivotal role in this process. Here, we studied the maturation status of DCs and expression of TonEBP in DCs exposed to hypertonic condition. METHODS: Murine bone marrow-derived DCs were generated in the presence of GM-CSF for 6 days, and then exposed to hypertonic media. We evaluated the functional capacities and maturation of DCs using flow cytometry, mixed lymphocyte reaction, and cytokine analysis. Also we investigated the expression of TonEBP in DCs cultured in variable hypertonic media. RESULTS: Mild hypertonicity made CD11c+ DCs to have up-regulation of CD40, 80, and 86. DCs exposed to 320 mOsm/kg media stimulated allogeneic T cell proliferation most effectively compared to DCs exposed to other tonic conditions. However, DCs exposed to 400 mOsm/kg media showed similar stimulatory capacities to isotonic control. Consistent with the phenotype changes, IL-1, 6, and TNF-alpha secretion increased in CD11c+ DCs exposed to mild hypertonic condition. Though we confirmed that TonEBP was expressed in CD11c+ DCs, the amount of upregulation was not dependent on the degree of hypertonicity. CONCLUSION: Our results suggest that hypertonicity enhances the maturation and activation of DCs.
Antigen-Presenting Cells ; Carrier Proteins ; Cell Proliferation ; Dendritic Cells ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; Interleukin-1 ; Lymphocyte Culture Test, Mixed ; Osmolar Concentration ; Phenotype ; T-Lymphocytes ; Tumor Necrosis Factor-alpha ; Up-Regulation

BACKGROUNDDendritic cells (DCs) are one of the most important antigen presenting cells in the human body, and DCs at various stages of maturation possess different or even opposite functions. The aim of this study was to investigate the influence of growth hormones on the functional status of cord blood-derived DCs encompassing immunophenotype, ability to excrete interleukin (IL)-12 and provoke autologous leukomonocyte.

METHODSMononuclear cells were isolated from fresh cord blood, with IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) used to induce and stimulate the mononuclear cells. Growth hormone at different concentrations was used to modify DCs, and then DCs morphology, number and growth status were observed. The immunophenotype of DCs was detected with a flow cytometer. The concentration of IL-12 in the DCs supernatant was determined by enzyme linked immunosorbent assay (ELISA) and DCs functional status was evaluated by autologous mixed lymphocyte reactions.

RESULTSMononuclear cells from cord blood can be differentiated into DCs by cytokine induction and growth hormone modification. With the increase in growth hormone concentrations (5 - 100 microg/L), the expression of DCs HLA-DR, CD1alpha, CD80 and CD83 were significantly increased (P < 0.05). The ability of DCs to secrete IL-12 was significantly improved (P < 0.05), and the ability of DCs to activate autologous lymphocytes was significantly enhanced (P < 0.05). Pegvisomant was able to ablate the effects of growth hormone on DCs.

CONCLUSIONSGrowth hormone may facilitate DCs induction and maturation, and improve the reproductive activity of autologous lymphocytes in a dose-dependent manner. Growth hormone may serve as a factor of modifying DCs to achieving maturity.

Antigens, CD ; metabolism ; B7-1 Antigen ; metabolism ; Cells, Cultured ; Dendritic Cells ; drug effects ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Growth Hormone ; pharmacology ; HLA-DR Antigens ; metabolism ; Humans ; Immunoglobulins ; metabolism ; Interleukin-12 ; metabolism ; Membrane Glycoproteins ; metabolism

The present study was aimed to investigate the pathways, by which IL-27 regulates the expression of adherent molecule Mac-1, chemotactic factor receptor fMLP-R and pro-inflammatory cytokine IL-1beta in human neutrophils. Highly purified human neutrophils were isolated from peripheral blood using Ficoll-Hypaque gradients centrifugation and erythrocyte lysis. The mRNA expression of IL-27 receptor components (WSX-1/TCCR and gp130) in human neutrophils was detected by reverse transcription polymerase chain reaction (RT-PCR). After incubation with IL-27 and specific inhibitors (p38 MAPK inhibitor SB203580, PI3K inhibitor LY294002 and ERK inhibitor U0126), the mRNA levels of fMLP-R and IL-1beta were determined by real time RT-PCR, and the adherent molecule Mac-1 expression in human neutrophils was determined by flow cytometry. The IL-1beta level in culture supernatant of human neutrophils was assayed by radioimmunoassay. The results showed that IL-27 receptor components (WSX-1/TCCR and gp130) were constitutively expressed in human neutrophils. IL-27 down-regulated Mac-1 expression in human neutrophils (p<0.05). After incubation with specific inhibitors, SB203580, not LY294002 and U0126, inhibited the down-regulation of Mac-1 expression by IL-27. However, IL-27 up-regulated the mRNA expression of fMLP-R and IL-1beta, and increased the release of IL-1beta (p<0.05). Interestingly, LY294002, not SB203580 and U0126, inhibited the up-regulation of fMLP-R and IL-1beta by IL-27. It is concluded that the IL-27 may regulate the expression of Mac-1, fMLP-R and IL-1beta in human neutrophils through p38 MAPK and PI3K signal pathways.
Butadienes ; pharmacology ; Chromones ; pharmacology ; Down-Regulation ; Humans ; Imidazoles ; pharmacology ; Interleukin-1beta ; metabolism ; Interleukins ; metabolism ; Macrophage-1 Antigen ; metabolism ; Morpholines ; pharmacology ; Neutrophils ; metabolism ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Pyridines ; pharmacology ; Receptors, Formyl Peptide ; metabolism ; Signal Transduction ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUNDLittle is known of the effects of hydrocortisone on cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and its counterreceptors (LFA-1, Mac-1) in acute pancreatitis (AP). We investigated the effects of prior treatment with hydrocortisone on the production of ICAM-1 and its counterreceptors (LFA-1 and Mac-1) in AP of rats to clarify the effect of hydrocortisone on induced acute pancreatitis.

METHODSAcute pancreatitis was induced by infusion of 5% chenodeoxycholic acid into the pancreatic duct, followed by ligation of pancreatic duct. Before induction of acute pancreatitis, rats were treated with hydrocortisone (n = 20) or 0.9% saline (n = 20). Blood and specimens from pancreas and lung were obtained from 5 rats from each treatment euthanized at 1 hour or 3 hours, 6 hours, 12 hours. Expression of ICAM-1 was assessed by immunohistochemistry and Western blot analysis of pancreas and lungs. The expression of LFA-1 and Mac-1 on neutrophils was detected by flow cytometer. The therapeutic effect of hydrocortisone was assessed from injuries to pancreas and lung.

RESULTSICAM-1 expression in the pancreas of hydrocortisone group was significantly less than in control group at 3 hours and 6 hours. In the lungs of hydrocortisone group, ICAM-1 expression was significantly less than in control group at 3 hours, 6 hours and 12 hours. The expression of LFA-1 and Mac-1 on neutrophils in blood increased significantly in control group over hydrocortisone group. Increased expression of ICAM-1, LFA-1 and Mac-1 preceded leukocyte infiltration. Compared to untreated animals with acute pancreatitis, rats pretreated with hydrocortisone had significantly reduced histological lung injury and output of ascitic fluid.

CONCLUSIONSPrior treatment with hydrocortisone before the induction of acute pancreatitis ameliorates pulmonary injury and the output of ascitic fluid and reduces the expression of ICAM-1 and its counterreceptors (LFA-1, Mac-1) in acute pancreatitis.

Acute Disease ; Amylases ; blood ; Animals ; Hydrocortisone ; pharmacology ; therapeutic use ; Intercellular Adhesion Molecule-1 ; analysis ; Lymphocyte Function-Associated Antigen-1 ; blood ; Macrophage-1 Antigen ; blood ; Pancreatitis ; blood ; drug therapy ; Rats ; Rats, Sprague-Dawley

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.
Cloning, Molecular ; Complement C3 ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Macrophage-1 Antigen ; genetics ; metabolism ; Receptors, Complement 3b ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

This study was aimed to evaluate the in vivo antitumor effect of genetically modified myeloma cell vaccine on human myeloma xenografts implanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human immune system was established in NOD/SCID mice by intraperitoneal injection of human peripheral blood lymphocytes (PBLs). After being inoculated subcutaneously with irradiated myeloma cell line sko-007, adenovirally transferred with GFP or p53, granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 genes, huPBL-NOD/SCID mice were challenged by subcutaneous injection of non-transferred sko-007 cells. The results indicated that Ad-p53/GM-CSF/B7-1-infected sko-007 cell vaccination significantly reduced local tumor growth compared with controls. Histopathological and immunohistochemical analysis showed that tumor tissues increasingly displayed diffuse necrosis, mainly caused by apoptosis, accompanied with significant fibroplasias and blood vessel hyperplasia, and human T cells infiltrated into the tumor tissues. It is concluded that transgenic p53, GM-CSF and B7-1 expression produces an immune response against myeloma cells and may be of therapeutic value for multiple myeloma in human being.
Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; genetics ; immunology ; Cancer Vaccines ; immunology ; Genes, p53 ; immunology ; Genetic Vectors ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Immunotherapy ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Multiple Myeloma ; immunology ; Neoplasm Transplantation

Platelet clearance has already been studied in physiological and pathological conditions and shown its occurrence mainly in the liver and the spleen. It is still not clear what mechanisms are responsible for recognition and removal of either aged or damaged platelets by the scavenging system. So study of the clearance mechanism will be useful to prolong the survival time of platelets in vivo. And it may be related to a new strategy to store platelets. This article focuses on the advances in studies of the clearance mechanism of transfused platelet concentrates, including roles of P-selectin, GPI balpha and its receptor Mac-1 in platelet clearance, and effect of endogenous metalloproteinase in platelet clearance.
Blood Platelets ; physiology ; Blood Preservation ; Cellular Senescence ; Humans ; Macrophage-1 Antigen ; physiology ; Metalloproteases ; physiology ; P-Selectin ; physiology ; Platelet Activation ; Platelet Glycoprotein GPIb-IX Complex ; physiology ; Platelet Transfusion