PURPOSE: Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1alpha (HIF-1alpha) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1alpha-induced invasive characteristics. MATERIALS AND METHODS: Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effect of hypoxia on HIF-1alpha, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1alpha and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1alpha, tissue analysis was done. RESULTS: Hypoxia induces HIF-1alpha expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1alpha via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1alpha with RNA interference suppressed hypoxia-induced HIF-1alpha and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1alpha. These were confirmed in the orthotopic FTC model. CONCLUSION: Hypoxia induced HIF-1alpha, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.
Adenocarcinoma, Follicular/*genetics/metabolism ; Animals ; Anoxia/*genetics ; Cadherins/genetics ; Epithelial-Mesenchymal Transition/*genetics ; Gene Expression Regulation, Neoplastic ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism ; Lymphokines ; Mice ; Neoplasm Invasiveness ; Phenotype ; Real-Time Polymerase Chain Reaction ; Signal Transduction/drug effects ; Thyroid Neoplasms/*genetics/metabolism ; Transcriptional Activation ; Twist Transcription Factor/*genetics/metabolism ; Vimentin/metabolism

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

The nomenclature "embryonic lymphoid tissue inducer (LTi) cell" reflects the fundamental role of the cell in secondary lymphoid tissue organization. In addition, it is equally important in primary lymphoid tissue development as it regulates central tolerance to self-antigens in the thymus. An adult LTi cell constitutively expresses two sets of tumor necrosis factor (TNF) family members, whereas its embryonic counterpart expresses only one. The first set is lymphotoxin (LT)alpha, LTbeta, and TNFalpha, which are essential for the secondary lymphoid organogenesis during embryogenesis and for maintaining an organized secondary lymphoid structure during adulthood. The second set is OX40- and CD30-ligands, which are critical for memory T cell generation. Adult LTi cells regulate adaptive immune responses by providing LTbetaR signals to stromal cells to maintain secondary lymphoid tissue structure, and determine adaptive immune responses by providing OX40 and CD30 survival signals to activated T cells in memory T cell generation. Along with the consideration of the roles of embryonic LTi cells in primary and secondary lymphoid tissues, this review highlights the roles of adult LTi cells in secondary lymphoid tissue function.
Adult ; Animals ; Humans ; Lymphoid Tissue/cytology/embryology/*immunology ; Lymphokines/immunology/metabolism ; T-Lymphocytes, Helper-Inducer/cytology/*immunology/metabolism ; Thymus Gland/cytology/embryology/*immunology

Animals ; Fatty Liver ; metabolism ; pathology ; Liver ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; Lymphokines ; metabolism ; Male ; Platelet-Derived Growth Factor ; metabolism ; Rats ; Rats, Sprague-Dawley

Vascular endothelial growth factor, VEGF, is essential for endothelial cell differentiation (vasculogenesis) and for the sprouting of new capillaries from preexisting vessels (angiogenesis). In addition, there is strong evidence that VEGF is a survival factor allowing the cells to survive and proliferate under conditions of extreme stress. Hypoxia is a key regulator of VEGF gene expression. Besides hypoxia, many cytokines, hormones and growth factors can up-regulate VEGF mRNA expression in various cell types. VEGF is present in the glomerulus of both the fetal and adult kidney. The VEGF produced by glomerular epithelial cell may be responsible for maintenance of the fenestrated phenotype of glomerular epithelial cells, thus facilitating the high rate of glomerular ultrafiltration. But there is little known about the role of VEGF in the tubule. VEGF is thought to be involved in many kinds of kidney diseases. Whereas VEGF has a beneficial role in the pathogenesis in some diseases, it does harmful action in others. Because VEGF is known to be associated with the pathogenesis of some diseases, such as diabetic nephropathy, renal tumor and polycystic kidney disease, the study about the role of VEGF is going to be a target for disease control. On the other hand, an attempt at enhancing the role of VEGF has to be made at diseases like several ARF models and experimental glomerulonephritis.
Animals ; Endothelial Growth Factors/genetics/*metabolism ; Gene Expression ; Human ; Intercellular Signaling Peptides and Proteins/genetics/*metabolism ; Kidney Diseases/*metabolism/physiopathology ; Kidney Glomerulus/*metabolism ; Kidney Tubules/*metabolism ; Lymphokines/genetics/*metabolism ; Protein Isoforms/genetics/metabolism ; Receptors, Vascular Endothelial Growth Factor/metabolism ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.

METHODSThe HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.

RESULTSHFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.

CONCLUSIONHFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.

Cell Adhesion ; Cell Cycle ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; metabolism ; Epidermis ; Fetus ; G1 Phase ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Keratinocytes ; cytology ; Keratins ; analysis ; Luminescent Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Microscopy, Fluorescence ; Plasmids ; genetics ; Stem Cells ; chemistry ; cytology ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

To investigate the relationship of the expression of vascular endothelial factor (VEGF) and matrix metalloproteinase-2 (MMP-2) with the recurrence and metastasis of hepatocellular carcinoma (HCC), the expression of VEGF and MMP-2 in HCC tissue(n = 50) and in normal liver tissue(n = 30) were examined by immunochemistry. The results showed that the positive rates of VEGF and MMP-2 in HCC tissue were 86% and 60% respectively, and in normal liver tissue were 53.3% and 30% respectively. The positive rates of VEGF and MMP-2 in HCC were significantly higher than those in normal liver tissue. The positive rates of VEGF and MMP-2 in HCC with intra- or extra-hepatic metastasis were higher than those of HCC without metastasis. VEGF and MMP-2 play important roles in the invasion and metastasis of HCC.
Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; secondary ; Endothelial Growth Factors ; biosynthesis ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Liver Neoplasms ; metabolism ; pathology ; Lymphokines ; biosynthesis ; Male ; Matrix Metalloproteinase 2 ; biosynthesis ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

To investigate the relationship of the expression of vascular endothelial growth factor (VEGF) and C-myc with the occurrence, development and metastasis of gallbladder carcinoma, the expression levels of VEGF and C-myc in gallbladder carcinoma tissue (n = 30) and in normal gallbladder tissue (n = 20) were examined by immunochemistry. Results show that the positive rates of VEGF and C-myc in gallbladder carcinoma tissue were 80% and 63.3% respectively, and 45% and 25% respectively in normal gallbladder tissue. The positive rates of VEGF and C-myc were significantly higher in gallbladder carcinoma than in normal gallbladder tissue. The expression of VEGF and C-myc in gallbladder carcinoma related to the metastasis of gallbladder carcinoma. VEGF and C-myc play important roles in the occurrence, development and metastasis of gallbladder carcinoma.
Adult ; Aged ; Endothelial Growth Factors ; biosynthesis ; physiology ; Female ; Gallbladder Neoplasms ; metabolism ; pathology ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; physiology ; Lymphokines ; biosynthesis ; physiology ; Male ; Middle Aged ; Neoplasm Metastasis ; Proto-Oncogene Proteins c-myc ; biosynthesis ; physiology ; 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