AIM: Macrophage migration inhibitory factor (MIF) was reported to inactivate p53 and play an essential role in the growth and angiogenesis of tumors that arise at sites of chronic inflammation. Gastric inflammation is...AIM: Macrophage migration inhibitory factor (MIF) was reported to inactivate p53 and play an essential role in the growth and angiogenesis of tumors that arise at sites of chronic inflammation. Gastric inflammation is a prerequisite for the development of gastric carcinoma (GC), which has recently been linked to Helicobacter pylori (H pylori) infection. This study aimed to investigate dinicopathological significance of MIF expression in GCs. METHODS: We selected 90 consecutive patients with GCs for investigation of the relation among MIF status, clinicopathological parameters, p53 expression and angiogenesis. MIF and p53 expression was assessed by immunohistochemistry as positive and negative groups. Tumor vascularity was evaluated by counting microvessel density on anti-CD34 stained sections. Expression status of MIF was correlated with determined dinicopathological data, p53 immunoreactivity and microvessel counts. RESULTS: Strong immunostainings of MIF were observed in the cytoplasm of cancerous cells in 40% (36/90) of cases but not in normal or metaplastic epithelia. There was no statistically significant correlation between MIF expression and age, gender, H pylori infection, tumor location, histological subtypes, lymph node metastasis or p53 expression. Early GC less frequently overexpressed MIF as compared to advanced GCs (4/20 vs 32/70, P= 0.04). A remarkably increased microvessel count was noted in GCs with MIF expression than those without MIF expression (55.1±30.1 vs 31.3±28.8, P= 0.0001). CONCLUSION: Our results suggest that expression of MIF may contribute to the progression and enhanced angiogenesis in a substantial portion of GCs.展开更多
AIM:To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27Kip1 in them,and further determine whether the e...AIM:To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27Kip1 in them,and further determine whether the effects are related to the PI3K/Akt signal transduction pathway. METHODS:Gastric cancer MGC-803 cells were cultured and then treated with 50 μg/L recombinant human MIF (rhMIF) with and without a PI3K inhibitor,LY294002 (25 μmol/L). MTT assay was used to detect the prolifer-ation of MGC-803 cells. Cell cycle was detected by flow cytometry. Expression of cyclin D1 and p27Kip1 mRNA was by reverse transcription-polymerase chain reaction. Protein expression of phosphorylated Akt (p-Akt),Akt,cyclin D1 and p27Kip1 was examined by immunocyto-chemistry and Western blotting. RESULTS:rhMIF signifi cantly stimulated the prolifera-tion of MGC-803 cells and cell cycle progression from G1 phase to S phase in a concentration-and time-de-pendent manner. After the MGC-803 cells were treated with rhMIF for 24 h,the expression of cyclin D1 was signifi cantly up-regulated compared with the cells not treated with rhMIF at both mRNA and protein levels(0.97 ± 0.02 vs 0.74 ± 0.01,P = 0.002; 0.98 ± 0.05 vs 0.69 ± 0.04,P = 0.003). The p27Kip1 was down-regulated but only statistically significant at the protein level. rhMIF significantly increased the expression of p-Akt,which reached the peak at 30 min,but did not affect the expression of Akt. However,LY294002 inhibited all the effects of rhMIF.CONCLUSION:Macrophage MIF increases the proliferation of gastric cancer cells,induces the expression of cyclin D1 at the transcriptional level and inhibits the expression of p27Kip1 at the post-transcriptional level via the PI3K/Akt pathway.展开更多
AIM: Helicobacter pylori (H pylori) is associated with increased gastric inflammatory and epithelial expression of macrophage migration inhibitory factor (MIF) and gastric epithelial cell proliferation. This study aim...AIM: Helicobacter pylori (H pylori) is associated with increased gastric inflammatory and epithelial expression of macrophage migration inhibitory factor (MIF) and gastric epithelial cell proliferation. This study aimed at determining whether H pylori directly stimulates release of MIF in monocytes, whether the cag pathogenicity island (PAI) is involved for this function, and whether MIF stimulated by H pylori increases gastric epithelial cell proliferation in vitro. METHODS: A cytotoxic wild-type H pylori strain (TN2), its three isogenic mutants (TN2Δcag,TN2ΔcagA and TN2ΔcagE) were co-cultured with cells of a human monocyte cell line, THP-1, for 24 h at different organism/ cell ratios. MIF in the supernatants was measured by an ELISA. Cells of a human gastric cancer cell line, MKN45, were then co-cultured with the supernatants, with and without monoclonal anti-MIF antibody for 24 h. The cells were further incubated for 12 h after addition of 3H-thymidine, and the levels of incorporation of 3H-thymidine were measured with a liquid scintillation counter. RESULTS: The wild-type strain and the isogenic mutants, TN2ΔcagA and TN2ΔcagE, increased MIF release at organism/cell ratios of 200/1 and 400/1, but not at the ratios of 50/1 and 100/1. However, the mutant TN2Δcag did not increase the release of MIF at any of the four ratios. 3H-thymidine readings for MKN-45 cells were significantly increased with supernatants derived from the wild-type strain and the mutants TN2ΔcagA and TN2ΔcagE, but not from the mutant TN2Δcag. Moreover, in the presence of monoclonal anti-MIF antibody, the stimulatory effects of the wild-type strain on cell proliferation disappeared. CONCLUSION: H pylori stimulates MIF release in monocytes, likely through its cag PAI, but not related to cagA or cagE. H pylori-stimulated monocyte culture supernatant increases gastric cell proliferation, which is blocked by anti-MIF antibody, suggesting that MIF plays an important role in H pylori-induced gastric epithelial cell proliferation.展开更多
We investigated the effects of mouse recombinant IL-4 on hematopoiesis in vitro and in vivo. IL-4 alone was found to be incapable of stimulating colony formation, but it inhibited both IL-3-and GM-CSF-induced colony f...We investigated the effects of mouse recombinant IL-4 on hematopoiesis in vitro and in vivo. IL-4 alone was found to be incapable of stimulating colony formation, but it inhibited both IL-3-and GM-CSF-induced colony formation by murine hematopoietic progenitor cells. In contrast, colony formation induced by G-CSF was enhanced in the presence of IL-4. We also studied the influence of IL-4 on hematopoietic reconstiution after allogeneic bone marrow transplantation in a murine medel, and found that IL-4 had significant inhibitory effects on neutrophil recovery and that neutrophil recovery accelerated by IL-3 and G-CSF was significantly suppressed by IL-4. The combination of IL-4 and GM-SF caused a significant decrease in the absolute number of neutrophils.展开更多
To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with...To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5′-TACGGATACAAGTAGCAC-3′; Sense, 5′-ATGCCTATGTTCATCGTG-3′; Missense, 5′-CTCTCAGACTCGATCTGT-3′. These phosphorothioate oligonucleotides were then transfected into cultured macrophages ( RAW264.7 ) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1?ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis. The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12?h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides. There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibit the expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.展开更多
Objective: To estimate directly phagocytic functions of the macrophages because of the importance in innate immunity, determine blood vessel density and re-innervation density which are basis of function. Methods: Eig...Objective: To estimate directly phagocytic functions of the macrophages because of the importance in innate immunity, determine blood vessel density and re-innervation density which are basis of function. Methods: Eighty adult Wistar rats were randonjy divided into experimental and control groups.The formers underwent splenotomy and a half splenie slice was transplanted into greater omentum. The latter only moved. After 6 months, examination was made as follows: ① After injection of 0.4% carbon particles by vein, spleme tissues were taken out at different times for estimating phagocytosis by light microscope. ② When splenic tissues had been intubated into left ventricle under total anesthesia, animals were perfused by formalin and India ink mixture suspension. Splenic tissues were taken out for making sections for measurement of area density of blood vessels.③ Immunohistochemical procedure was used for detecting neuropeptide Y(NPY). Results: Phagocytie functions had no difference between two groups, hut the area density of blood vessels and NPY-positive fibers re-dued (P<0.01) in experimental group. Conclusion:Autotransplanted splenic tissues show good innate immunity though regeneration of blood vessels and nerves do not reach normal level.展开更多
基金Supported by the Grants From National Science Council (NSC2314-B002-122,123,124), Executive Yuan, Taiwan, China
文摘AIM: Macrophage migration inhibitory factor (MIF) was reported to inactivate p53 and play an essential role in the growth and angiogenesis of tumors that arise at sites of chronic inflammation. Gastric inflammation is a prerequisite for the development of gastric carcinoma (GC), which has recently been linked to Helicobacter pylori (H pylori) infection. This study aimed to investigate dinicopathological significance of MIF expression in GCs. METHODS: We selected 90 consecutive patients with GCs for investigation of the relation among MIF status, clinicopathological parameters, p53 expression and angiogenesis. MIF and p53 expression was assessed by immunohistochemistry as positive and negative groups. Tumor vascularity was evaluated by counting microvessel density on anti-CD34 stained sections. Expression status of MIF was correlated with determined dinicopathological data, p53 immunoreactivity and microvessel counts. RESULTS: Strong immunostainings of MIF were observed in the cytoplasm of cancerous cells in 40% (36/90) of cases but not in normal or metaplastic epithelia. There was no statistically significant correlation between MIF expression and age, gender, H pylori infection, tumor location, histological subtypes, lymph node metastasis or p53 expression. Early GC less frequently overexpressed MIF as compared to advanced GCs (4/20 vs 32/70, P= 0.04). A remarkably increased microvessel count was noted in GCs with MIF expression than those without MIF expression (55.1±30.1 vs 31.3±28.8, P= 0.0001). CONCLUSION: Our results suggest that expression of MIF may contribute to the progression and enhanced angiogenesis in a substantial portion of GCs.
基金Supported by Grant from Hunan Provincial Science and Technology Department (2008 FJ 3088), China
文摘AIM:To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27Kip1 in them,and further determine whether the effects are related to the PI3K/Akt signal transduction pathway. METHODS:Gastric cancer MGC-803 cells were cultured and then treated with 50 μg/L recombinant human MIF (rhMIF) with and without a PI3K inhibitor,LY294002 (25 μmol/L). MTT assay was used to detect the prolifer-ation of MGC-803 cells. Cell cycle was detected by flow cytometry. Expression of cyclin D1 and p27Kip1 mRNA was by reverse transcription-polymerase chain reaction. Protein expression of phosphorylated Akt (p-Akt),Akt,cyclin D1 and p27Kip1 was examined by immunocyto-chemistry and Western blotting. RESULTS:rhMIF signifi cantly stimulated the prolifera-tion of MGC-803 cells and cell cycle progression from G1 phase to S phase in a concentration-and time-de-pendent manner. After the MGC-803 cells were treated with rhMIF for 24 h,the expression of cyclin D1 was signifi cantly up-regulated compared with the cells not treated with rhMIF at both mRNA and protein levels(0.97 ± 0.02 vs 0.74 ± 0.01,P = 0.002; 0.98 ± 0.05 vs 0.69 ± 0.04,P = 0.003). The p27Kip1 was down-regulated but only statistically significant at the protein level. rhMIF significantly increased the expression of p-Akt,which reached the peak at 30 min,but did not affect the expression of Akt. However,LY294002 inhibited all the effects of rhMIF.CONCLUSION:Macrophage MIF increases the proliferation of gastric cancer cells,induces the expression of cyclin D1 at the transcriptional level and inhibits the expression of p27Kip1 at the post-transcriptional level via the PI3K/Akt pathway.
基金Supported by a Competitive Earmarked Research Grant from the Research Grants Council of Hong Kong Special Administrative Region, China HKU7318/01M
文摘AIM: Helicobacter pylori (H pylori) is associated with increased gastric inflammatory and epithelial expression of macrophage migration inhibitory factor (MIF) and gastric epithelial cell proliferation. This study aimed at determining whether H pylori directly stimulates release of MIF in monocytes, whether the cag pathogenicity island (PAI) is involved for this function, and whether MIF stimulated by H pylori increases gastric epithelial cell proliferation in vitro. METHODS: A cytotoxic wild-type H pylori strain (TN2), its three isogenic mutants (TN2Δcag,TN2ΔcagA and TN2ΔcagE) were co-cultured with cells of a human monocyte cell line, THP-1, for 24 h at different organism/ cell ratios. MIF in the supernatants was measured by an ELISA. Cells of a human gastric cancer cell line, MKN45, were then co-cultured with the supernatants, with and without monoclonal anti-MIF antibody for 24 h. The cells were further incubated for 12 h after addition of 3H-thymidine, and the levels of incorporation of 3H-thymidine were measured with a liquid scintillation counter. RESULTS: The wild-type strain and the isogenic mutants, TN2ΔcagA and TN2ΔcagE, increased MIF release at organism/cell ratios of 200/1 and 400/1, but not at the ratios of 50/1 and 100/1. However, the mutant TN2Δcag did not increase the release of MIF at any of the four ratios. 3H-thymidine readings for MKN-45 cells were significantly increased with supernatants derived from the wild-type strain and the mutants TN2ΔcagA and TN2ΔcagE, but not from the mutant TN2Δcag. Moreover, in the presence of monoclonal anti-MIF antibody, the stimulatory effects of the wild-type strain on cell proliferation disappeared. CONCLUSION: H pylori stimulates MIF release in monocytes, likely through its cag PAI, but not related to cagA or cagE. H pylori-stimulated monocyte culture supernatant increases gastric cell proliferation, which is blocked by anti-MIF antibody, suggesting that MIF plays an important role in H pylori-induced gastric epithelial cell proliferation.
文摘We investigated the effects of mouse recombinant IL-4 on hematopoiesis in vitro and in vivo. IL-4 alone was found to be incapable of stimulating colony formation, but it inhibited both IL-3-and GM-CSF-induced colony formation by murine hematopoietic progenitor cells. In contrast, colony formation induced by G-CSF was enhanced in the presence of IL-4. We also studied the influence of IL-4 on hematopoietic reconstiution after allogeneic bone marrow transplantation in a murine medel, and found that IL-4 had significant inhibitory effects on neutrophil recovery and that neutrophil recovery accelerated by IL-3 and G-CSF was significantly suppressed by IL-4. The combination of IL-4 and GM-SF caused a significant decrease in the absolute number of neutrophils.
文摘To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5′-TACGGATACAAGTAGCAC-3′; Sense, 5′-ATGCCTATGTTCATCGTG-3′; Missense, 5′-CTCTCAGACTCGATCTGT-3′. These phosphorothioate oligonucleotides were then transfected into cultured macrophages ( RAW264.7 ) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1?ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis. The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12?h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides. There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibit the expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.
基金Supported by the Foundation for Sci & Tech Research Project of Chongqing, China (2003)
文摘Objective: To estimate directly phagocytic functions of the macrophages because of the importance in innate immunity, determine blood vessel density and re-innervation density which are basis of function. Methods: Eighty adult Wistar rats were randonjy divided into experimental and control groups.The formers underwent splenotomy and a half splenie slice was transplanted into greater omentum. The latter only moved. After 6 months, examination was made as follows: ① After injection of 0.4% carbon particles by vein, spleme tissues were taken out at different times for estimating phagocytosis by light microscope. ② When splenic tissues had been intubated into left ventricle under total anesthesia, animals were perfused by formalin and India ink mixture suspension. Splenic tissues were taken out for making sections for measurement of area density of blood vessels.③ Immunohistochemical procedure was used for detecting neuropeptide Y(NPY). Results: Phagocytie functions had no difference between two groups, hut the area density of blood vessels and NPY-positive fibers re-dued (P<0.01) in experimental group. Conclusion:Autotransplanted splenic tissues show good innate immunity though regeneration of blood vessels and nerves do not reach normal level.
