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72 Pirfenidone blunts induction of bleomycin induced genes associated with idiopathic pulmonary fibrosis in mice
Abstracts / Cytokine 43 (2008) 243–262 Acute renal failure is an abrupt decrease in renal function. Interleukin (IL)-10 inhibits ischemic and cisplatin-induced acute renal failure. We aimed to determine whether IL-20 affects renal tubular epithelial cells and is associated with acute renal failure. We analyzed the expression of IL-20 and its receptor (R) in the kidneys of rats with HgCl2-induced acute renal failure. Reverse transcription-PCR showed upregulated IL-20 and its receptors and immunohistochemical staining showed strongly expressed IL-20 protein in proximal tubular epithelial cells. We analyzed human proximal tubular epithelial (HK-2) cells, which expressed both IL-20 and its receptors. IL-20 specifically induced mitochondriadependent apoptosis by activating caspase 9 in HK-2 cells. IL-20 also activated c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2, the downstream signals implicated in the apoptosis of HK-2 cells. Furthermore, IL-20 upregulated the transcripts of transforming growth factor (TGF)-b1, a critical mediator of renal injury. In hypoxic HK-2 cells, IL-20 and IL-22R1 transcripts increased, and IL-20 upregulated IL-1b transcripts. In vivo study further demonstrated that anti-IL-20 antibody reduced the expression of TGF-b1, and IL-1bf nand the number of damaged tubular cells in the kidneys of rats with acute renal failure. We concluded that IL-20 may be involved in the injury of renal epithelial cells in acute renal failure. doi:10.1016/j.cyto.2008.07.110
70 Plasma concentration sE-selectin in patients with postoperative infectious complications from gastrointestinal surgery for stomach cancer Zelenskiy Alexey 1, Kazakov Sergey 2, 1 State postgraduate medical education of the Defense Ministry of Russian Federation, Moscow, Russia, 2 Main Military Clinical Hospital name of N.N. Burdenko, Moscow, Russia Background: We studied the change in plasma concentration sE-selectin in the perioperative period in patients diagnosed with stomach cancer. The study is to evaluate the predictive capabilities sE-selectin in the development of infectious complications. Patients and Methods: We studied plasma 64 patients were treated in the period from 2003 to 2007. Diagnosis carried flow cytometry method (Coulter Epix XL-MCL), using sets of ‘‘Multiplex” (Bender Medsystems). Analysis of the data was carried out using a set of statistical programs SPSS 13,0. Results: The following patterns were identified: patients with a concentration of sE-selectin >40 pg/ml in the preoperative period, the probability of development of infectious complications was 2.5 times higher (p < 0,01), with concentrations of sE-selectin >72,2 pg/ml in the second postoperative day, the likelihood of development of infectious complications was 3 times higher (p < 0001), with concentrations of sE-selectin >88,1 pg/ml in the third postoperative day-5 times higher (p < 0001). Conclusions: It was found that the study plasma concentration sE-selectin in the perioperative period in patients with stomach cancer reveals risk of development of infectious complications in the early postoperative period. doi:10.1016/j.cyto.2008.07.111
71 Tumor cells induce interferon responsive genes in primary endothelial cells in a polar fashion Claudia Nemeth, Herwig P. Moll, Harald Freudenthaler, Anna Zommer, Christine Brostjan, Department of Surgery—Research Laboratories, Medical University of Vienna, Austria In tumor angiogenesis, signals derived from cancer cells activate endothelial cells to proliferate, migrate and form new blood vessels for tumor supply and expansion. Using a transwell co-culture system of human microvascular endothelial cells (ECs) and tumor cells we aimed to reproduce and characterize these interactions. We found the induction of interferon responsive genes such as IFIT-1 and ISG15 in ECs to be elicited by certain colon or breast cancer cell lines (HT-29, BT-549). Unlike type I interferon, the tumor-derived stimulus was of high molecular weight and restricted to EC activation from the apical side. Nevertheless, the type I IFN receptor IFNAR was required for an efficient EC response. Since expression of IFIT1 and ISG15 mRNA peaked at 8–14 h and protein levels reached a maximum around 20 h of stimulation, a possible feedback signal by autocrine IFN production might be involved. Considering a potential stimulus originating from microbial contaminants of cancer cells, the cell lines were repeatedly tested but found negative for any viral or bacterial infections. Alternatively, activation of interferon responsive genes via toll-like receptor (TLR) signaling could occur by other cancer derived factors such as fragments of the extracellular matrix component hyaluronic acid. The TLR-related concomitant activation of transcription factor NF-jB was, however, not observed in our setting. In the pursuit to identify the tumor derived stimulus capable of inducing interferon-responsive genes in ECs in
253
a polar fashion, we are currently analyzing the tumor culture supernatant by fractionation, gel filtration, and shotgun proteomics. doi:10.1016/j.cyto.2008.07.112
72 Pirfenidone blunts induction of bleomycin induced genes associated with idiopathic pulmonary fibrosis in mice Milton W. Taylor 1, Takuma Tsukahara 2, Osman N. Ozes 2, Jena Derrick 2, Sarah K. Stevens 2, Lawrence M. Blatt 2, 1 Department of Biology, Indiana University, Bloomington, IN, USA, 2 InterMune Inc. Brisbane, CA, USA Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease, limited to lungs, of unknown etiology that results in significant morbidity and mortality. Several mediators, including TGF-beta, are likely to contribute to the development of fibrosis in IPF. An anti-fibrotic investigational drug candidate, pirfenidone (PFD) has been used in the treatment of this disease with some success. To understand the biological pathways that are influenced by pirfenidone treatment, we analyzed DNA microarrays from lungs of C57/Bl6 mice that were exposed with intratracheal bleomycin followed by continuous treatment with PFD for 14 days At the end of the study, lungs were excised, protein prepared for Western blots and RNA extracted for microarray analysis (Affymetrix). Bleomycin alone induced 483 gene probes and down regulated 133 gene probes (>2.0 fold, p < 0.001). The number of genes modified was drastically reduced in the presence of bleomycin and PFD (127 induced and 10 down regulated). PFD alone had no effect on gene induction or down regulation. A large number of genes associated with extracellular matrix formation, including collagen, fibronectin and elastin were blunted by PFD. Metalloproteinases (MMP) 2, 8, 11, 12, and 14 were induced by bleomycin, and all were blunted. Chemokine CCL9 was also highly induced and reduced by PFD but other chemokines were not affected. TGFb2 and TGFb3 were slightly induced and lower in the presence of PFD. The transcription factor Wisp1 was highly induced by bleomycin and reduced by PFD. This gene encodes a member of the WNT1 inducible signaling pathway protein subfamily, which belongs to the connective tissue growth factor (CTGF) family. We conclude that PFD has an effect on several groups of fibrosis-associated genes that are induced by bleomycin. doi:10.1016/j.cyto.2008.07.113
73 Multiplex immunoassays for the quantification of rhesus and cynomolgus monkey cytokines Mary M. Brodey, Nina Liu, Jimin Wang, Kevin Reagan, Invitrogen Corporation, Camarillo, CA, USA Non-human primates, such as rhesus monkey (Macaca mulatta) and cynomolgus monkey (Macaca fascicularis), possess a high degree of gene homology with humans, as well as similar biochemistry and metabolism. These animals are therefore useful in a number of areas of biomedical research including neuroscience, neuroendocrinology, reproductive physiology, and cardiovascular physiology. Non-human primates serve as important models for human infectious diseases such as AIDS and tuberculosis. Non-human primates are also used routinely in target validation and toxicity studies in drug and vaccine development. To facilitate studies involving non-human primates, Invitrogen has recently developed a series of assays for the LuminexÒ xMAPTM platform that permit the detection and quantification of cytokines in rhesus and cynomolgus monkey. Markers include: G-CSF, IFN-c, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40/p70, IL-17, MIP-1a, MIP-1b, MCP-1, RANTES, and TNF-a. These assays permit the development of single-analyte assays, or may be assembled into multiplexes for simultaneous measurement of multiple analytes. The utility of the assays was demonstrated by determining cytokine profiles obtained with LPS- or PMA plus A23187-stimulated peripheral blood mononuclear cells (PBMCs) from rhesus monkeys and cynomolgus monkeys. Linearity of dilution experiments with tissue culture medium and serum produced R2 values of 0.98 and greater, while sample recovery routinely fell between 80% and 120%. Intra-assay and inter-assay precision was 610%. doi:10.1016/j.cyto.2008.07.114
74 Inhibition by AT-1001 of epithelial tight junction permeability caused by cytokines from PT-gliadin stimulated human PBMC Malarvizhi Durai, Neil Poloso, Kelly Kitchens, Robert Somerville, Rosa Carrasco, Shobha Gopalakrishnan, Amir Tamiz, Niranjan Pandey, Sefik S. Alkan, ALBA Therapeutics, Baltimore, MD 21201, USA
70 Plasma concentration sE-selectin in patients with postoperative infectious complications from gastrointestinal surgery for stomach cancer Zelenskiy Alexey 1, Kazakov Sergey 2, 1 State postgraduate medical education of the Defense Ministry of Russian Federation, Moscow, Russia, 2 Main Military Clinical Hospital name of N.N. Burdenko, Moscow, Russia Background: We studied the change in plasma concentration sE-selectin in the perioperative period in patients diagnosed with stomach cancer. The study is to evaluate the predictive capabilities sE-selectin in the development of infectious complications. Patients and Methods: We studied plasma 64 patients were treated in the period from 2003 to 2007. Diagnosis carried flow cytometry method (Coulter Epix XL-MCL), using sets of ‘‘Multiplex” (Bender Medsystems). Analysis of the data was carried out using a set of statistical programs SPSS 13,0. Results: The following patterns were identified: patients with a concentration of sE-selectin >40 pg/ml in the preoperative period, the probability of development of infectious complications was 2.5 times higher (p < 0,01), with concentrations of sE-selectin >72,2 pg/ml in the second postoperative day, the likelihood of development of infectious complications was 3 times higher (p < 0001), with concentrations of sE-selectin >88,1 pg/ml in the third postoperative day-5 times higher (p < 0001). Conclusions: It was found that the study plasma concentration sE-selectin in the perioperative period in patients with stomach cancer reveals risk of development of infectious complications in the early postoperative period. doi:10.1016/j.cyto.2008.07.111
71 Tumor cells induce interferon responsive genes in primary endothelial cells in a polar fashion Claudia Nemeth, Herwig P. Moll, Harald Freudenthaler, Anna Zommer, Christine Brostjan, Department of Surgery—Research Laboratories, Medical University of Vienna, Austria In tumor angiogenesis, signals derived from cancer cells activate endothelial cells to proliferate, migrate and form new blood vessels for tumor supply and expansion. Using a transwell co-culture system of human microvascular endothelial cells (ECs) and tumor cells we aimed to reproduce and characterize these interactions. We found the induction of interferon responsive genes such as IFIT-1 and ISG15 in ECs to be elicited by certain colon or breast cancer cell lines (HT-29, BT-549). Unlike type I interferon, the tumor-derived stimulus was of high molecular weight and restricted to EC activation from the apical side. Nevertheless, the type I IFN receptor IFNAR was required for an efficient EC response. Since expression of IFIT1 and ISG15 mRNA peaked at 8–14 h and protein levels reached a maximum around 20 h of stimulation, a possible feedback signal by autocrine IFN production might be involved. Considering a potential stimulus originating from microbial contaminants of cancer cells, the cell lines were repeatedly tested but found negative for any viral or bacterial infections. Alternatively, activation of interferon responsive genes via toll-like receptor (TLR) signaling could occur by other cancer derived factors such as fragments of the extracellular matrix component hyaluronic acid. The TLR-related concomitant activation of transcription factor NF-jB was, however, not observed in our setting. In the pursuit to identify the tumor derived stimulus capable of inducing interferon-responsive genes in ECs in
253
a polar fashion, we are currently analyzing the tumor culture supernatant by fractionation, gel filtration, and shotgun proteomics. doi:10.1016/j.cyto.2008.07.112
72 Pirfenidone blunts induction of bleomycin induced genes associated with idiopathic pulmonary fibrosis in mice Milton W. Taylor 1, Takuma Tsukahara 2, Osman N. Ozes 2, Jena Derrick 2, Sarah K. Stevens 2, Lawrence M. Blatt 2, 1 Department of Biology, Indiana University, Bloomington, IN, USA, 2 InterMune Inc. Brisbane, CA, USA Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease, limited to lungs, of unknown etiology that results in significant morbidity and mortality. Several mediators, including TGF-beta, are likely to contribute to the development of fibrosis in IPF. An anti-fibrotic investigational drug candidate, pirfenidone (PFD) has been used in the treatment of this disease with some success. To understand the biological pathways that are influenced by pirfenidone treatment, we analyzed DNA microarrays from lungs of C57/Bl6 mice that were exposed with intratracheal bleomycin followed by continuous treatment with PFD for 14 days At the end of the study, lungs were excised, protein prepared for Western blots and RNA extracted for microarray analysis (Affymetrix). Bleomycin alone induced 483 gene probes and down regulated 133 gene probes (>2.0 fold, p < 0.001). The number of genes modified was drastically reduced in the presence of bleomycin and PFD (127 induced and 10 down regulated). PFD alone had no effect on gene induction or down regulation. A large number of genes associated with extracellular matrix formation, including collagen, fibronectin and elastin were blunted by PFD. Metalloproteinases (MMP) 2, 8, 11, 12, and 14 were induced by bleomycin, and all were blunted. Chemokine CCL9 was also highly induced and reduced by PFD but other chemokines were not affected. TGFb2 and TGFb3 were slightly induced and lower in the presence of PFD. The transcription factor Wisp1 was highly induced by bleomycin and reduced by PFD. This gene encodes a member of the WNT1 inducible signaling pathway protein subfamily, which belongs to the connective tissue growth factor (CTGF) family. We conclude that PFD has an effect on several groups of fibrosis-associated genes that are induced by bleomycin. doi:10.1016/j.cyto.2008.07.113
73 Multiplex immunoassays for the quantification of rhesus and cynomolgus monkey cytokines Mary M. Brodey, Nina Liu, Jimin Wang, Kevin Reagan, Invitrogen Corporation, Camarillo, CA, USA Non-human primates, such as rhesus monkey (Macaca mulatta) and cynomolgus monkey (Macaca fascicularis), possess a high degree of gene homology with humans, as well as similar biochemistry and metabolism. These animals are therefore useful in a number of areas of biomedical research including neuroscience, neuroendocrinology, reproductive physiology, and cardiovascular physiology. Non-human primates serve as important models for human infectious diseases such as AIDS and tuberculosis. Non-human primates are also used routinely in target validation and toxicity studies in drug and vaccine development. To facilitate studies involving non-human primates, Invitrogen has recently developed a series of assays for the LuminexÒ xMAPTM platform that permit the detection and quantification of cytokines in rhesus and cynomolgus monkey. Markers include: G-CSF, IFN-c, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40/p70, IL-17, MIP-1a, MIP-1b, MCP-1, RANTES, and TNF-a. These assays permit the development of single-analyte assays, or may be assembled into multiplexes for simultaneous measurement of multiple analytes. The utility of the assays was demonstrated by determining cytokine profiles obtained with LPS- or PMA plus A23187-stimulated peripheral blood mononuclear cells (PBMCs) from rhesus monkeys and cynomolgus monkeys. Linearity of dilution experiments with tissue culture medium and serum produced R2 values of 0.98 and greater, while sample recovery routinely fell between 80% and 120%. Intra-assay and inter-assay precision was 610%. doi:10.1016/j.cyto.2008.07.114
74 Inhibition by AT-1001 of epithelial tight junction permeability caused by cytokines from PT-gliadin stimulated human PBMC Malarvizhi Durai, Neil Poloso, Kelly Kitchens, Robert Somerville, Rosa Carrasco, Shobha Gopalakrishnan, Amir Tamiz, Niranjan Pandey, Sefik S. Alkan, ALBA Therapeutics, Baltimore, MD 21201, USA