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S2049 Primary Study in Animal Model of Sporadic Colorectal Adenomas
to up-regulation of Bcl-2 (by PCR and western blot) in these cells isolated from C57Black/ 6 and from NOD/SCID mice. Conclusions: CD74 is expressed on C57Black/6 CEC and serves as survival receptor. These results may have implications on colorectal cancer research.
the anti-inflammatory IL-4 and IL-10 cytokines. CONCLUSIONS: Our results suggest that SATB1 expression plays an important role in CRC and seems to be associated with an aggressive phenotype. SATB1 correlates with high expression of TNF alpha, which mediates the inflammatory processes in tumor invasion, angiogenesis and metastasis. On the other hand, SATB1 also influences expression of anti-inflammatory IL-4 and IL-10 which in turn is known to lead to escape from cancer immune-surveillance. Future studies targeting expression of SATB1 may lead to a novel approach to inhibit aggressive behavior of these cancers.
AGA Abstracts
S2048 Claudin-1 Protein, Which Is Tight Junction Specific Molecule, Is a Major Factor Involved in the Tumorigenesis of Colorectal Cancer Tetsushi Kinugasa, Yoshito Akagi, Kazuo Shirouzu
S2051
Background: The molecular and morphological alterations of the tight junctions in colorectal cancer (CRC) are still poorly understood. The possible involvement of Claudin-1 (CL-1), one of the major tight junctional proteins (TJPs), was investigated in the tumorigenesis of colorectal cancer. Patients and Methods: CRC tissues specimens and their paired normal mucosa were analyzed to determine whether the expression of CL-1 correlates with the clinicopathological factors and to determine the role of CL-1 in the alteration of tight junctions during tumorigenesis. Adenocarcinoma tissue and paired normal mucosa specimens were resected from surgical specimens of CRC patients. Results: The expression of CL-1 at the mRNA and protein levels were analyzed in 41 cases. The expression was found to increase in comparison to that in normal tissues specimens. The mRNA levels of CL-1 were correlated with tumor depth but not with the preoperative carcinoembryonic antigen (CEA) serum level. When T84 cells, a human colon cancer cell line, was transfected with the CL1 gene, the CL-1 overexpressing cells grew as aggregates in contrast to the monolayer formation of the parental cells. In addition, trypsin-treated CL-1 overexpressing cells aggregated more easily than did the parental cells. Conclusion: These observations suggested that CL-1 plays a pivotal role in the regulation of the cellular morphology and behavior in the colonic epithelium. CL-1 protein may therefore be one of the major factors involved in the tumorigenesis of CRC.
Stromal Cell EGFR Enhances Colon Cancer Cell Growth in Tumor Xenografts Urszula Dougherty, Dario Cerasi, Ieva Taylor, Reba Mustafi, Hongyan Zhu, John Hart, Loren J. Joseph, David W. Threadgill, Alessandro Fichera, Marc Bissonnette Background: Epidermal growth factor receptors (EGFR) are over-expressed in 20-70% of human colorectal cancers. This receptor is expressed in both malignant epithelial cells and tumor stromal cells, including myofibroblasts. We have shown that EGFR stimulation increases colon cancer cell proliferation and induces cyclooxygenase-2 (Cox-2) in colonic myofibroblasts In Vitro. Cox-2 plays a key role in colonic tumorigenesis. Colonic myofibroblasts are an important source of Cox-2 in the stroma of premalignant tumors. To assess the role of stromal cell EGFR in tumor growth, we compared tumor xenografts in recipient mice with wild type or homozygous Waved-2 (Wa2) EGFR. The inhibitory Wa2 Egfr mutation reduces receptor kinase activity by 90% in assays In Vitro. Methods: For this study we prepared immuno-tolerant Rag1-/- mice that were wild type (Wt) for Egfr or homozygous for Wa2 Egfr by breeding Rag1-/- mice with Egfr Wt/Wa2 heterozygous mice. Rag1-/- and Wa2 mice were both on a C57Bl6 background. Double heterozygous F1 mice were mated to generate double homozygous Rag1- Wa2 mice (with EGFR deficient stroma) and control Rag1-/- mice with Wt EGFR. HCT116 colon cancer cells (5 x 10^6 cells) were implanted subcutaneously into the flank of Rag1-/- Wt EGFR and Rag1-/- Wa2/Wa2 mice. Tumors were harvested 3 wks later and tumor weights and dimensions measured. Proliferation marker Ki67, apoptosis marker, cleaved caspase-3 and angiogenesis marker nestin-1 were assessed by immunostaining and quantified by computer assisted image analysis. EGFR signals including phospho-active EGFR (pEGFR) and pERK and targets, cyclin D1 and Cox2 were measured by quantitative Western blotting. Results: Subcutaneous tumors were significantly smaller in Wa2/Wa2 mice compared to tumors in mice with Wt Egfr (0.26±0.03 vs. 0.63±0.06g, p<0.05). Compared to tumors in mice with Wt Egfr, Ki67 staining was reduced by 67%, and angiogenesis significantly decreased by 40%, whereas apoptosis as assessed by cleaved caspase-3 was 2.9-fold higher in tumors from mice with Wa2 Egfr (p<0.05). Western blotting revealed >5-fold increases in β catenin expression, and EGFR signals (pEGFR and pERK), and >3-fold up-regulated cyclin D1 and Cox-2 in tumors growing in mice with Wt EGFR, compared to Wa2 mice (p<0.05). Summary: Increases in β-catenin and activation of EGFR signals in tumor xenografts require EGFR-regulated paracrine signals from stromal cells. Stromal cell EGFR signals promote tumor growth by enhancing colon cancer cell proliferation and angiogenesis, while inhibiting apoptosis. These studies demonstrate for the first time that stromal cell EGFR regulates colon cancer cell growth.
S2049 Primary Study in Animal Model of Sporadic Colorectal Adenomas Yaopeng Zhang, Yumin Li Background: There are two animal models for researches of colorectal tumors, colorectal cancer model and aberrant crypt foci model. Both models can not fulfill the requirements of short experiment periods and sufficient tissues for molecular research at the same time. Many studies have shown the deactivation or partial activation of Peroxisome proliferators activated receptor-γ (PPAR-γ) may play an important role in colorectal cancer. This experiment was designed to establish an experimental animal model of sporadic colorectal adenoma with an antagonist of PPAR-γ based on traditional DMH-induced colorectal cancer model which can satisfy these two requirements at the same time. Methods: Thirty seven male Sprague-Dawley rats were randomly grouped into four groups, negative control group (9 rats), Group DMH (10 rats), Group DMH+GW9662 (9 rats) and Group DMH+GW9662+Pioglitazone (9 rats). Dimethylhydrazine (DMH) as the carcinogenic agent was injected intraperitoneally at a dose of 120mg/kg body weight at week 0, and was given a second shot subcutaneously at a dose of 20mg/kg body weight at week 2. GW9662, as a complete antagonist of PPAR-γ, was injected intravenously once a week at a dose of 0.1mg/ kg body weight and with a triple dose at the initial. Pioglitazone, as an agonist of PPAR-γ, was mixed into animal feeds at a dose of 44ppm. GW9662 and pioglitazone lasted to week 12 and all rats were sacrificed by euthanasia then. Half of the distal colorectum was taken and immersed in formalin at 4°C overnight, and then recorded the polyps by anatomic microscope when stained by methylene blue. All polyps and some flatten risen mucosa were verified by histology. Results: Five rats died at the early stage of the experiment without adenoma formation. There was no adenoma in negative control group. One adenoma was found in Group DMH, and the incidence of adenoma was 12.5 % (1/8), the load of adenoma was 0.125 (1/8). In Group DMH +GW9662, 16 adenomas were found in 7 rats, and the incidence of adenoma was 87.5% (7/8), and the load of adenoma was 2.0 (16/8). There were 5 adenoma in 2 rats, and the incidence and load of adenoma in Group DMH+GW9662+Pioglitazone were 28.6% (2/7), 0.714(5/7) respectively. Conclusion: The combination of DMH and GW9662 can induce rat colorectal adenoma efficiently in a short experiment period with plenty adenoma tissue for further mechanism research. This animal model can be used in colorectal adenoma and pre-cancerous lesions related researches and could overcome some defects of colorectal carcinoma model and ACF model.
S2052 The Behavior of Serum Matrix Metalloproteinase -2,-7 and -9 in the Normal Mucosa-Adenoma-Colorectal Carcinoma Sequence László Herszényi, István Hritz, István Gábor, Ferenc Sipos, Gábor Lakatos, Zsolt Tulassay Background: Matrix metalloproteinases (MMP-s) play a part in colorectal cancer (CRC) invasion and metastasis. Strong expression of many MMPs has been related to poor survival in CRC patients. However, the behavior of serum MMPs has scarcely been investigated in the normal mucosa-adenoma-adenocarcinoma sequence of the colon. Methods: The MMP2, MMP-7 and MMP-9 serum levels were determined in 14 patients with CRC, 8 patients with colorectal tubulovillous adenoma and low-grade dysplasia, 10 patients with inflammatory bowel disease (IBD) and 8 tumor-free control patients by ELISA technique. Statistical analysis with one-way ANOVA was performed. P value of <0.05 was considered significant. Results: MMP-2 and MMP-9 serum antigen levels (mean values+/-standard deviation, ng/ ml) were significantly higher in CRC and adenomas compared to controls (MMP-2: 176.8+/35.2, 185.1+/-31.9 and 147.1+/-21.2, respectively, P<0.05; MMP-9: 645.9+/-149.8, 667.39+/-91.8 and 451.1+/-67.6, respectively, P<0.01). MMP-9 was also higher in patients with IBD compared to controls (637.1+/-154.6 vs 451.1+/-67.6, P<0.05). Significantly higher MMP-7 levels were observed in patients with CRC and adenomas compared with IBD and controls (5.3+/-2.0, 5.2+/-1.8, 3.6+/-2.5 and 2.5+/-0.8, respectively, P<0.05). Conclusions: We demonstrate that antigen levels of MMP-2,-7 and -9 were significantly higher in blood samples from patients with CRC and adenomas compared to the controls, suggesting that MMPs may have a crucial role not only in the invasive process of cancer, but also in the progression of colorectal premalignant adenomas into CRC. Our results confirm previous results obtained in colorectal tissues that MMP-9 could also contribute to the inflammatory processes in IBD.
S2050 SATB1 Influences Expression of Cytokines in Colorectal Cell Lines and Is Associated with a More Aggressive Phenotype Agnieszka M. Rygiel, Akueni L. Davelaar, Francesca Milano, Paul Fockens, Kausilia K. Krishnadath BACKROUND: SATB1 is a nuclear protein well known for its crucial role in regulating gene expression during the differentiation and activation of T cells. This makes it a key player in the regulation of the cytokine expression profile of T-cells in the immune system. Very recently, it has been shown that SATB1 (Han HJ et al., Nature 2008 Mar) is also involved in tumor growth and metastasis of breast cancer by orchestrating the expression of large number of genes involved in growth, proliferation, angiogenesis and metastasis. AIM: Here, we addressed the role of SATB1 in colorectal cancer (CRC) and particularly its association with expression of pro- and anti-inflammatory cytokines. MATERIAL and METHODS: The CRC cell lines that we examined for expression of SATB1 by RT-PCR, Western Blot and immunohistochemistry (IHC), included the highly metastatic HT-29, HCT-116 and RKO, and the non metastatic SW48 and SW480. The supernatant from the cells lines was examined for Th1/Th2 cytokines expression by Cytometric bead Array (CBA). RESULTS: We found that SATB1 is expressed in two of the highly metastatic cell lines HT-29 and RKO, whereas HCT-116 and the non metastatic SW48 and SW480 were negative. Further, we showed that both SATB1 expressing cell lines, RKO and HT-29, release high levels of the proinflammatory TNF-alpha. Interestingly, in HT-29 but not RKO we detected expression of
AGA Abstracts
S2053 A New Method for Detection and Monitoring of Colonic Carcinogenesis and Angiogenesis in a Novel Mouse Model Valentin Becker, Florian Greten, Irina Kerle, Roland M. Schmid, Alexander Meining Background: Probe-based confocal endomicroscopy (pCLE) enables In Vivo histopathology and determination of micro vessel density. We aimed to combine this method with miniaturized colonoscopy for evaluation of cancer development and assessment of vascularization in a novel mouse model. Material and Methods: We crossed “floxed” p53 mice with villin-Cre transgenic mice. Colonic cancers were provoked after repetitively injection of azoxymethane (AOM). A total of 8 mice were examined before and 36 days after AOM injection. An endoscope with a 3mm outer diameter (Karl Storz, Germany) was introduced into the colon.
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the anti-inflammatory IL-4 and IL-10 cytokines. CONCLUSIONS: Our results suggest that SATB1 expression plays an important role in CRC and seems to be associated with an aggressive phenotype. SATB1 correlates with high expression of TNF alpha, which mediates the inflammatory processes in tumor invasion, angiogenesis and metastasis. On the other hand, SATB1 also influences expression of anti-inflammatory IL-4 and IL-10 which in turn is known to lead to escape from cancer immune-surveillance. Future studies targeting expression of SATB1 may lead to a novel approach to inhibit aggressive behavior of these cancers.
AGA Abstracts
S2048 Claudin-1 Protein, Which Is Tight Junction Specific Molecule, Is a Major Factor Involved in the Tumorigenesis of Colorectal Cancer Tetsushi Kinugasa, Yoshito Akagi, Kazuo Shirouzu
S2051
Background: The molecular and morphological alterations of the tight junctions in colorectal cancer (CRC) are still poorly understood. The possible involvement of Claudin-1 (CL-1), one of the major tight junctional proteins (TJPs), was investigated in the tumorigenesis of colorectal cancer. Patients and Methods: CRC tissues specimens and their paired normal mucosa were analyzed to determine whether the expression of CL-1 correlates with the clinicopathological factors and to determine the role of CL-1 in the alteration of tight junctions during tumorigenesis. Adenocarcinoma tissue and paired normal mucosa specimens were resected from surgical specimens of CRC patients. Results: The expression of CL-1 at the mRNA and protein levels were analyzed in 41 cases. The expression was found to increase in comparison to that in normal tissues specimens. The mRNA levels of CL-1 were correlated with tumor depth but not with the preoperative carcinoembryonic antigen (CEA) serum level. When T84 cells, a human colon cancer cell line, was transfected with the CL1 gene, the CL-1 overexpressing cells grew as aggregates in contrast to the monolayer formation of the parental cells. In addition, trypsin-treated CL-1 overexpressing cells aggregated more easily than did the parental cells. Conclusion: These observations suggested that CL-1 plays a pivotal role in the regulation of the cellular morphology and behavior in the colonic epithelium. CL-1 protein may therefore be one of the major factors involved in the tumorigenesis of CRC.
Stromal Cell EGFR Enhances Colon Cancer Cell Growth in Tumor Xenografts Urszula Dougherty, Dario Cerasi, Ieva Taylor, Reba Mustafi, Hongyan Zhu, John Hart, Loren J. Joseph, David W. Threadgill, Alessandro Fichera, Marc Bissonnette Background: Epidermal growth factor receptors (EGFR) are over-expressed in 20-70% of human colorectal cancers. This receptor is expressed in both malignant epithelial cells and tumor stromal cells, including myofibroblasts. We have shown that EGFR stimulation increases colon cancer cell proliferation and induces cyclooxygenase-2 (Cox-2) in colonic myofibroblasts In Vitro. Cox-2 plays a key role in colonic tumorigenesis. Colonic myofibroblasts are an important source of Cox-2 in the stroma of premalignant tumors. To assess the role of stromal cell EGFR in tumor growth, we compared tumor xenografts in recipient mice with wild type or homozygous Waved-2 (Wa2) EGFR. The inhibitory Wa2 Egfr mutation reduces receptor kinase activity by 90% in assays In Vitro. Methods: For this study we prepared immuno-tolerant Rag1-/- mice that were wild type (Wt) for Egfr or homozygous for Wa2 Egfr by breeding Rag1-/- mice with Egfr Wt/Wa2 heterozygous mice. Rag1-/- and Wa2 mice were both on a C57Bl6 background. Double heterozygous F1 mice were mated to generate double homozygous Rag1- Wa2 mice (with EGFR deficient stroma) and control Rag1-/- mice with Wt EGFR. HCT116 colon cancer cells (5 x 10^6 cells) were implanted subcutaneously into the flank of Rag1-/- Wt EGFR and Rag1-/- Wa2/Wa2 mice. Tumors were harvested 3 wks later and tumor weights and dimensions measured. Proliferation marker Ki67, apoptosis marker, cleaved caspase-3 and angiogenesis marker nestin-1 were assessed by immunostaining and quantified by computer assisted image analysis. EGFR signals including phospho-active EGFR (pEGFR) and pERK and targets, cyclin D1 and Cox2 were measured by quantitative Western blotting. Results: Subcutaneous tumors were significantly smaller in Wa2/Wa2 mice compared to tumors in mice with Wt Egfr (0.26±0.03 vs. 0.63±0.06g, p<0.05). Compared to tumors in mice with Wt Egfr, Ki67 staining was reduced by 67%, and angiogenesis significantly decreased by 40%, whereas apoptosis as assessed by cleaved caspase-3 was 2.9-fold higher in tumors from mice with Wa2 Egfr (p<0.05). Western blotting revealed >5-fold increases in β catenin expression, and EGFR signals (pEGFR and pERK), and >3-fold up-regulated cyclin D1 and Cox-2 in tumors growing in mice with Wt EGFR, compared to Wa2 mice (p<0.05). Summary: Increases in β-catenin and activation of EGFR signals in tumor xenografts require EGFR-regulated paracrine signals from stromal cells. Stromal cell EGFR signals promote tumor growth by enhancing colon cancer cell proliferation and angiogenesis, while inhibiting apoptosis. These studies demonstrate for the first time that stromal cell EGFR regulates colon cancer cell growth.
S2049 Primary Study in Animal Model of Sporadic Colorectal Adenomas Yaopeng Zhang, Yumin Li Background: There are two animal models for researches of colorectal tumors, colorectal cancer model and aberrant crypt foci model. Both models can not fulfill the requirements of short experiment periods and sufficient tissues for molecular research at the same time. Many studies have shown the deactivation or partial activation of Peroxisome proliferators activated receptor-γ (PPAR-γ) may play an important role in colorectal cancer. This experiment was designed to establish an experimental animal model of sporadic colorectal adenoma with an antagonist of PPAR-γ based on traditional DMH-induced colorectal cancer model which can satisfy these two requirements at the same time. Methods: Thirty seven male Sprague-Dawley rats were randomly grouped into four groups, negative control group (9 rats), Group DMH (10 rats), Group DMH+GW9662 (9 rats) and Group DMH+GW9662+Pioglitazone (9 rats). Dimethylhydrazine (DMH) as the carcinogenic agent was injected intraperitoneally at a dose of 120mg/kg body weight at week 0, and was given a second shot subcutaneously at a dose of 20mg/kg body weight at week 2. GW9662, as a complete antagonist of PPAR-γ, was injected intravenously once a week at a dose of 0.1mg/ kg body weight and with a triple dose at the initial. Pioglitazone, as an agonist of PPAR-γ, was mixed into animal feeds at a dose of 44ppm. GW9662 and pioglitazone lasted to week 12 and all rats were sacrificed by euthanasia then. Half of the distal colorectum was taken and immersed in formalin at 4°C overnight, and then recorded the polyps by anatomic microscope when stained by methylene blue. All polyps and some flatten risen mucosa were verified by histology. Results: Five rats died at the early stage of the experiment without adenoma formation. There was no adenoma in negative control group. One adenoma was found in Group DMH, and the incidence of adenoma was 12.5 % (1/8), the load of adenoma was 0.125 (1/8). In Group DMH +GW9662, 16 adenomas were found in 7 rats, and the incidence of adenoma was 87.5% (7/8), and the load of adenoma was 2.0 (16/8). There were 5 adenoma in 2 rats, and the incidence and load of adenoma in Group DMH+GW9662+Pioglitazone were 28.6% (2/7), 0.714(5/7) respectively. Conclusion: The combination of DMH and GW9662 can induce rat colorectal adenoma efficiently in a short experiment period with plenty adenoma tissue for further mechanism research. This animal model can be used in colorectal adenoma and pre-cancerous lesions related researches and could overcome some defects of colorectal carcinoma model and ACF model.
S2052 The Behavior of Serum Matrix Metalloproteinase -2,-7 and -9 in the Normal Mucosa-Adenoma-Colorectal Carcinoma Sequence László Herszényi, István Hritz, István Gábor, Ferenc Sipos, Gábor Lakatos, Zsolt Tulassay Background: Matrix metalloproteinases (MMP-s) play a part in colorectal cancer (CRC) invasion and metastasis. Strong expression of many MMPs has been related to poor survival in CRC patients. However, the behavior of serum MMPs has scarcely been investigated in the normal mucosa-adenoma-adenocarcinoma sequence of the colon. Methods: The MMP2, MMP-7 and MMP-9 serum levels were determined in 14 patients with CRC, 8 patients with colorectal tubulovillous adenoma and low-grade dysplasia, 10 patients with inflammatory bowel disease (IBD) and 8 tumor-free control patients by ELISA technique. Statistical analysis with one-way ANOVA was performed. P value of <0.05 was considered significant. Results: MMP-2 and MMP-9 serum antigen levels (mean values+/-standard deviation, ng/ ml) were significantly higher in CRC and adenomas compared to controls (MMP-2: 176.8+/35.2, 185.1+/-31.9 and 147.1+/-21.2, respectively, P<0.05; MMP-9: 645.9+/-149.8, 667.39+/-91.8 and 451.1+/-67.6, respectively, P<0.01). MMP-9 was also higher in patients with IBD compared to controls (637.1+/-154.6 vs 451.1+/-67.6, P<0.05). Significantly higher MMP-7 levels were observed in patients with CRC and adenomas compared with IBD and controls (5.3+/-2.0, 5.2+/-1.8, 3.6+/-2.5 and 2.5+/-0.8, respectively, P<0.05). Conclusions: We demonstrate that antigen levels of MMP-2,-7 and -9 were significantly higher in blood samples from patients with CRC and adenomas compared to the controls, suggesting that MMPs may have a crucial role not only in the invasive process of cancer, but also in the progression of colorectal premalignant adenomas into CRC. Our results confirm previous results obtained in colorectal tissues that MMP-9 could also contribute to the inflammatory processes in IBD.
S2050 SATB1 Influences Expression of Cytokines in Colorectal Cell Lines and Is Associated with a More Aggressive Phenotype Agnieszka M. Rygiel, Akueni L. Davelaar, Francesca Milano, Paul Fockens, Kausilia K. Krishnadath BACKROUND: SATB1 is a nuclear protein well known for its crucial role in regulating gene expression during the differentiation and activation of T cells. This makes it a key player in the regulation of the cytokine expression profile of T-cells in the immune system. Very recently, it has been shown that SATB1 (Han HJ et al., Nature 2008 Mar) is also involved in tumor growth and metastasis of breast cancer by orchestrating the expression of large number of genes involved in growth, proliferation, angiogenesis and metastasis. AIM: Here, we addressed the role of SATB1 in colorectal cancer (CRC) and particularly its association with expression of pro- and anti-inflammatory cytokines. MATERIAL and METHODS: The CRC cell lines that we examined for expression of SATB1 by RT-PCR, Western Blot and immunohistochemistry (IHC), included the highly metastatic HT-29, HCT-116 and RKO, and the non metastatic SW48 and SW480. The supernatant from the cells lines was examined for Th1/Th2 cytokines expression by Cytometric bead Array (CBA). RESULTS: We found that SATB1 is expressed in two of the highly metastatic cell lines HT-29 and RKO, whereas HCT-116 and the non metastatic SW48 and SW480 were negative. Further, we showed that both SATB1 expressing cell lines, RKO and HT-29, release high levels of the proinflammatory TNF-alpha. Interestingly, in HT-29 but not RKO we detected expression of
AGA Abstracts
S2053 A New Method for Detection and Monitoring of Colonic Carcinogenesis and Angiogenesis in a Novel Mouse Model Valentin Becker, Florian Greten, Irina Kerle, Roland M. Schmid, Alexander Meining Background: Probe-based confocal endomicroscopy (pCLE) enables In Vivo histopathology and determination of micro vessel density. We aimed to combine this method with miniaturized colonoscopy for evaluation of cancer development and assessment of vascularization in a novel mouse model. Material and Methods: We crossed “floxed” p53 mice with villin-Cre transgenic mice. Colonic cancers were provoked after repetitively injection of azoxymethane (AOM). A total of 8 mice were examined before and 36 days after AOM injection. An endoscope with a 3mm outer diameter (Karl Storz, Germany) was introduced into the colon.
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