Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naive or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.
Animals ; Cell Proliferation ; Cells, Cultured ; Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors/metabolism ; Interleukin-10/*biosynthesis ; *Lymphocyte Activation ; Lymphokines/pharmacology ; Mesenchymal Stem Cells/cytology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Interleukin-10/metabolism ; T-Lymphocytes/cytology/*immunology/metabolism ; Tryptophan/analogs & derivatives/pharmacology

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naive or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.
Animals ; Cell Proliferation ; Cells, Cultured ; Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors/metabolism ; Interleukin-10/*biosynthesis ; *Lymphocyte Activation ; Lymphokines/pharmacology ; Mesenchymal Stem Cells/cytology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Interleukin-10/metabolism ; T-Lymphocytes/cytology/*immunology/metabolism ; Tryptophan/analogs & derivatives/pharmacology

OBJECTIVETo explore the response of nasal mucosa epithelial cells to hypoxia in terms of formation of nasal polyps (NP).

METHODSEpithelial cells of NP and inferior turbinate (IT) were cultured serum-free under normal oxygen and hypoxic circumstances with stimulation of IL-1 beta and TNF alpha. The vascular endothelial growth factor (VEGF) mRNA and VEGF protein levels of the cultured cells were detected using in situ hybridization and ELISA, respectively.

RESULTSThe expression of VEGF mRNA was significantly higher in epithelial cells of NP than in IT exposed to pro-inflammatory cytokines or hypoxia (P < 0.01). VEGF levels were higher in NP epithelial cells than those of IT (P < 0.01) under hypoxia.

CONCLUSIONVEGF-induced by hypoxia is very important for the early stages of forming polyps.

Cell Hypoxia ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Interleukin-1 ; pharmacology ; Lymphokines ; genetics ; Nasal Mucosa ; metabolism ; Nasal Polyps ; etiology ; metabolism ; RNA, Messenger ; analysis ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

To investigate the effect of arsenic trioxide (As(2)O(3)) on vascular endothelial growth factor (VEGF) expression in different courses of chronic myeloid leukemia (CML), VEGF level was measured with ELISA in the cultural supernatants of bone marrow mononuclear cells from CML patients. The results showed that supernatants of cultured bone marrow cells from 35 CML patients (20 chronic, 8 accelerated and 7 blast crisis phases) contained significantly higher VEGF levels (649.16 +/- 382.20 pg/ml, 560.27 +/- 409.14 pg/ml and 587.18 +/- 415.28 pg/ml, respectively) than that in 15 normal control samples (152.16 +/- 150.09 pg/ml; P < 0.01), but no significant differences were found in VEGF levels among different phases of CML. The bone marrow cells treated with As(2)O(3) (5 x 10(-6)mol/L) for 72 hours resulted in significant reduction of VEGF levels (down to 396.66 +/- 257.47 pg/ml, 363.42 +/- 239.85 pg/ml and 407.47 +/- 219.38 pg/ml, respectively) (P < 0.05). In conclusion, abnormal high expression of VEGF plays a role in the pathogenetic course of CML and it is probably an additional anticancer mechanism for As(2)O(3) to inhibit VEGF expression of leukemic cells.
Adolescent ; Adult ; Aged ; Arsenicals ; pharmacology ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; Child ; Culture Media, Conditioned ; chemistry ; Down-Regulation ; drug effects ; Endothelial Growth Factors ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; pathology ; Lymphokines ; metabolism ; Male ; Middle Aged ; Oxides ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.
Animals ; Antigens, CD34 ; immunology ; Binding Sites ; genetics ; CHO Cells ; Cell Division ; drug effects ; physiology ; Colony-Forming Units Assay ; Cricetinae ; Endothelial Growth Factors ; pharmacology ; Fetal Blood ; cytology ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Glycoproteins ; genetics ; pharmacology ; physiology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Interleukin-3 ; pharmacology ; Lymphokines ; pharmacology ; Membrane Proteins ; genetics ; RNA ; genetics ; metabolism ; Receptor, Notch1 ; Receptors, Cell Surface ; Recombinant Proteins ; isolation & purification ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; Transcription Factors ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the value of application of recombinant human VEGF to accelerate flap viability in a rat model of non-ischemic prefabricated flap.

METHODSPrefabricated Flaps were created in 48 SD rats. An autologous tail artery loop was anastomosed to the femoral artery and vein, and implanted subcutaneously in the lower abdomen. Flaps were divided into four groups of 12 each. At the time of loop implantation, the control groups received 0.9% NaCl (Control 1) and 16% (V/W) polyvinyl alcohol (PVA) solution (Control 2). The treatment groups received VEGF in 0.9% NaCl (treatment 1) and VEGF in PVA (treatment 2). In each group, a 3 cm x 4 cm flap nurtured by the tail artery pedicle was elevated and resutured into place after 3, 4 and 5 weeks. The percentage of surviving skin of each flap was determined by planimetry 7 days after flap elevation.

RESULTSMean skin survival areas at 3, 4, and 5 weeks were 1%, 0%, 10% in control; 0%, 16%, 25% in control 2; 3.57%, 39.13%, 75.00% in treatment 1; 8.13%, 41.98%, 58.41% in treatment 2. VEGF significantly improved flap survival by 5 weeks (P < 0.05).

CONCLUSIONThese results suggest VEGF can accelerate maturation of prefabricated flaps.

Animals ; Endothelial Growth Factors ; pharmacology ; Female ; Lymphokines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Surgical Flaps ; physiology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVESTo investigate mechanism for the increasing level of serum vascular endothelial growth factor(VEGF) in tumour patients during radiotherapy and the inhibitory action of the antisense oligodeoxynucleotide (AS-ODN) to the expression of VEGF protein by radiotherapy in the prostate cancer cell line (PC3M).

METHODSTo observe the changes of serum VEGF in the prostate cancer patients during radiotherapy dynamically and the inhibitory action of the antisense oligodeoxynucleotide to the expression of VEGF by radiotherapy in PC3M.

RESULTSThe changes of serum VEGF in three patients receiving radiotherapy had been observed continuously. The levels of serum VEGF began to increase when the patients received radiotherapy and rised up to peak value after fifteen days, then declined to the range of pre-radiotherapy. Irradiating the PC3M cells with X-rays significantly increased the VEGF expression and secretion. The expression of VEGF protein in the group treated by VEGF AS-ODNs and X-ray irradiation decreased significantly than the group treated only by X-ray irradiation.

CONCLUSIONSThe induction of VEGF protein expression by X-ray irradiation in tumor cells may result in the increasing of the VEGF in the prostate cancer patients during radiotherapy and the induction can be blocked by VEGF AS-ODNs.

DNA, Antisense ; pharmacology ; Endothelial Growth Factors ; antagonists & inhibitors ; blood ; genetics ; Gene Expression ; drug effects ; radiation effects ; Humans ; Intercellular Signaling Peptides and Proteins ; blood ; genetics ; Lymphokines ; antagonists & inhibitors ; blood ; genetics ; Male ; Prostatic Neoplasms ; blood ; pathology ; Radiotherapy ; adverse effects ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo screen for the inhibitor of vascular endothelial growth factor (VEGF) 165 from random peptide library.

METHODSPositive phage clones were rescued after two rounds of panning and competitive elution. Its affinity activity to KDR was monitored through ELISA, immunohistochemical method, Chicken CAM assay and MTT.

RESULTSFive specific binding positive target molecule phage clones were obtained which were able to bind to cells whose surface had high KDR, among which, clone 3 and 13 could effectively block the vascularization of the chorioallantoic membrane of chick embryo, but they were not inhibitive on the proliferation of high KDR expression cells.

CONCLUSIONThe peptides, being the inhibitors of VEGF, may be useful in the treatment of cancers.

Animals ; Binding Sites ; Endothelial Growth Factors ; antagonists & inhibitors ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Lymphokines ; antagonists & inhibitors ; metabolism ; Peptide Library ; Peptides ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the killing effect of herpes simplex virus thymidine kinase gene expression actuated by the promoter of the vascular endothelial growth factor gene on human hepatocellular tumor cells under hypoxic condition.

METHODSRecombinant adenoviral vectors, AdVEGF-tk and AdVEGF-GFP, were constructed with HSV-tk or GFP under the control of VEGF promoter through AdEasy system. Then GFP expression in hepatoma cell line HepG2 and normal liver cell line L02 transfected with AdVEGF-GFP were observed under fluorescence microscope, and the sensitivity to GCV of the AdVEGF-tk-transfected cells under normoxia or hypoxia condition were monitored by MTT method.

RESULTSGFP expression actuated by VEGF promoter was detected in sporadic L02 cells, but in almost all HepG2 cells after transfected with AdVEGF-GFP. With GCV at 10 micro g/ml and MOI at 100, L02 cells were insensitive to GCV under oxic condition, but more than 70% L02 cells were killed under hypoxic condition. Moreover, HepG2 cells infected with AdVEGF-tk showed the increased GCV sensitivity under hypoxia (over 80% killed) as compared with normoxia (over 60% killed) conditions.

CONCLUSIONHypoxia enhances the GCV sensitivity of human hepatocellular tumor cells infected with recombinant AdVEGF-tk under the control of VEGF promoter.

Adenoviridae ; genetics ; Carcinoma, Hepatocellular ; pathology ; Endothelial Growth Factors ; genetics ; Gene Expression ; drug effects ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Hypoxia ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver Neoplasms ; pathology ; Lymphokines ; genetics ; Oxygen ; pharmacology ; Promoter Regions, Genetic ; physiology ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo observe the effect of human vascular endothelial growth factor (VEGF) fragment (3 approximately 4 exon) in E. coli on anti-angiogenesis.

METHODSThrough RT-PCR amplification, endonuclease cut and DNA sequence analysis identification, hVEGF fragment cDNA was inserted into E. coli expression vector pTrcHis2A. The prokaryotic expression plasmid pTrcHis2A/VEGF(3 approximately 4) was constructed and transformed into TOP10F.

RESULTSAfter 8hr isopropy-beta-D-thiogalactoside (IPTG) induction, VEGF fragment was expressed in 15% of total proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expressed protein was highly antigenic and specific. The VEGF fragment was further purified by affinity, which could inhibit HUVEC proliferation and neovascularization of the chick chorioallantoic membrane.

CONCLUSIONVEGF fragment is anti-angiogenetic, which may potentially be used in oncologico-biological targeting therapy.

Angiogenesis Inhibitors ; isolation & purification ; pharmacology ; Cloning, Molecular ; Endothelial Growth Factors ; genetics ; isolation & purification ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; drug effects ; Genetic Vectors ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; isolation & purification ; pharmacology ; Isopropyl Thiogalactoside ; pharmacology ; Lymphokines ; genetics ; isolation & purification ; pharmacology ; Peptide Fragments ; genetics ; isolation & purification ; pharmacology ; Plasmids ; genetics ; Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors