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106 Strategy for a robust preclinical human assay to assess potential for cytokine release syndrome with therapeutic antibodies
260
Abstracts / Cytokine 43 (2008) 243–262
promoter reveals several STAT binding sites, suggesting that ps20 expression may be regulated transcriptionally by IFN. Using different truncated ps20 promoter constructs, we present data to describe the effects of IFN on that transcriptional regulation of ps20.
sensorineural hearing loss associated with inflammatory mediators such as TNF-a commonly found in the middle ear cleft of otitis media patients is still unclear from this experiment.
doi:10.1016/j.cyto.2008.07.143
doi:10.1016/j.cyto.2008.07.145
103 Experimental assessment of photodynamic therapy with 5,10,15,20-tetrakis(methoxyphenyl)-porphyrins in rat walker tumor Adriana G. Filip 1, Simona V. Clichici 1, Adriana V. Muresan 1, Claudia Gherman 2, Doina Daicoviciu 1, Rodica M. Ion 3, Simina Dreve 4, Nicoleta Decea 1, Remus Moldovan 1, 1 Department of Physiology, ‘‘Iuliu Hatieganu” University of Medicine and Pharmacy ClujNapoca, Romania, 2 Surgery Department, Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, 3 National R&D Institute of Chemistry and Petrochemistry—ICECHIM, Bucharest, Romania, 4 National R&D Institute of Isotopic and Molecular Technologies, Cluj-Napoca, Romania
105 Cytokine evaluation of seven different respiratory infections Matthew C. Groll, Peter Bellos, Quansys Biosciences, Logan, Utah USA. Positive samples from Influenza A, B, Parainfluenza 1,2,3
PDT-mediated oxidative stress elicits direct tumor cell damage as well as microvascular injury. In addition to this direct killing process, tumor eradication also arises from an acute inflammatory response featured by an increased level of various mediators in the PDT-treated tumor area (IL-1 b, G-CSF, IL- 8, and MIP-2). To improve this treatment new photosensitizes are being synthesized and tested. Our study evaluates the effects of PDT with 5,10,15,20-tetrakis- (methoxyphenyl)-porphyrins (TMPP) and zinc-5, 10,15,20-tetrakis-(methoxyphenyl)-porphyrins (Zn-TMPP) upon the serical and tumoral levels of ROS, IL4 and TNFa on Wistar male rats bearing Walker carcinosarcoma 256. Rats were randomly divided into groups as follows: group 1—no treatment, group 2—the tumors were only irradiated, group 3 were given TMPP 10 mg/kg b.w. and irradiated, group 4—were given Zn-TMPP 10 mg/kg b.w. and irradiated, group 5—were given 5-aminolevulinic acid (5-ALA) 250-mg/kg b.w. and irradiated. Laser light (at fluency rate 100 J/cm2 and pulse frequency rate 10 kHz) at 685 nm was delivered to tumors for 15 min following 24 h after drug administration. The MDA levels in serum at 24 h after treatment were lower than those obtained with 5-ALA (TMPP: 1.37 ± 0.13: ZnTMPP: 1.44 ± 0. 21 nmoles/mg protein vs. 3.37 ± 0.70 nmoles/mg protein, p < 0,01). Regarding the levels of carbonylated proteins PDT with TMPP and ZnTMPP showed significantly increased levels compared to the control group (TMPP: 1.08 ± 0.39; ZnTMPP: 1.03 ± 0.22 vs. control: 0.61 ± 0.07 nmoles/mg protein; p < 0,01). These results suggest that the oxygen reactive species are involved in PDT action. Effect of PDT with TMPP and ZnTMPP on the immune system involves enhanced cytokine secretion (IL4 and TNFa), which may account for the subsequent tumor eradication by PDT. doi:10.1016/j.cyto.2008.07.144
104 Role for tumor necrosis factor-alpha (TNF-a) in hearing loss and in aquaporin-4 (AQP-4) expression Min Kyo Jung 1, Song E. Kim 1, Sang W. Yeo 1, Steven K. Juhn 2, 1 Department of Otolaryngology-HNS, School of Medicine, The Catholic University of Korea, Seoul, Korea, 2 Department of Otolaryngology, University of Minnesota Medical School, Minneapolis, MN, USA Sensorineural hearing loss is rare, but one of the serious sequelae of otitis media. Although the mechanism has not yet been clarified, inflammatory process of middle ear has been known to be propagated to inner ear possibly through the round window membrane, and to bring about sensorineural hearing loss. After inflammatory mediators present or produced in the middle ear cleft such as endotoxin, lipopolysachharides, nitric oxide, reactive oxygen species, arachidonic acid metabolites and cytokines enter the inner ear, they can disturb inner ear homeostasis and function, and trigger ongoing inflammatory process there. The dysfunction of the inner ear, especially hearing loss, seems to be brought about by direct ototoxicity to the hair cells or by disturbances in homeostasis associated with potassium ion recycling. In addition to the role of gap junctions and ion pumps that actively transporting ions in the cochlea, aquaporins are known to be exist in the cochlea as a passive water channel. Among the subtypes of aquaporins exist in the cochlea, aquaporin 4 (AQP4) is the most abundant and concentrate in the supporting cells neighboring hair cells, and seem to have function to cope with osmotic gradient during the excitation of hair cells with influx of potassium ions. For AQP4 knock out mice have been reported to have hearing loss, AQP4 seems to be related to the hearing loss somehow when the inner ear homeostasis is disturbed. We demonstrated that TNF-a, a principal proinflammatory cytokine in the pathogenesis of otitis media, induced sensorineural hearing losses in rats experimentally. TNF receptor type I (TNFRI) proved to be expressed in the organ of Corti and lateral ligament in the rat cochlear. TNF-a may induce sensorineural hearing loss through the mechanisms such as induction of nitric oxide synthase and the disturbances in the physiologic potassium ion recycling in the cochlear. However, the role for AQP4 expression in the pathogenesis of
Adenovirus and respiratory syncytial virus (RSV) were tested for 16 different cytokines to evaluate the presence or absences of cytokines during acute infection. Two samples from each infection were taken and tested in duplicate using the Quansys Biosciences Human Cytokine Screen Plus Array. This panel contains 50 nl spots of capture antibodies to the following cytokines; IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-23, IFNc, TNFa and TNFb. The samples were prepared in HAMA treated buffer. The samples were then loaded into a 96 well plate and incubated for 1 hour at room temperature. The plate was then washed and incubated with detection antibody mix with antibodies for each of the 16 assays. Post incubation, the plate was washed again and incubated with streptavidin-HRP and incubated for 15 min. The plate was then washed and chemiluminescent substrate was added. The plate was imaged for 1 min using a Quansys Imager. The Flu A, Flu B and RSV samples showed a typical inflammatory response with a high expression of IL-6 and IL-8. The Para 1 samples showed both IL-6 and IL-8 responses with one sample showing high values for IL-1a, IL-1b, IL-5 and TNFa. Para 3 samples showed similar IL-1a, IL-6 and IL-8 elevated values. Yet, one sample showed high levels of IL-5, IL12p70 and IL-13. The Adenovirus samples showed similar cytokine expression levels, IL-1b, IL-6, IL-8, IL-10, IFNc and TNFa. Many of the cytokine responses were expected such as IL-1b, IL-6 and IL-8 as seen in typical inflammation conditions. However, it was interesting to observe the disparity between samples of the same disease conditions ranging with very different cytokine profiles such as was observed in the Parainfluenza 1, 2 and 3 samples. doi:10.1016/j.cyto.2008.07.146
106 Strategy for a robust preclinical human assay to assess potential for cytokine release syndrome with therapeutic antibodies Ram Achuthanandam, Peter J. Bugelski, George Treacy, Renold J. Capocasale, Toxicology and Investigative Pharmacology, Centocor R&D, Radnor, PA, USA Cytokine release reactions are associated with administration of some therapeutic antibodies. These reactions vary in clinical significance from mild fever and nausea to severe reactions including hypotension, tachycardia and multi-organ failure. When these symptoms are anticipated they can be managed by prophylactic treatment with a wide variety of compounds. Here we present a system where we can assess potential for cytokine release syndrome (CRS) when administering therapeutic antibodies using polychromatic flow cytometry and a pattern recognition methodology. The antibody of interest is presented to human whole blood using solid phase beads (24 h incubation). A transport inhibitor is added to block cytokine secretion from within the cells during the last phase of the incubation. After incubation, the cells are washed, fixed and stained for a 7-color panel interrogating cell surface phenotype (CD19: B-cell, CD2: T-cells and NK cells and CD15: monocytes and granulocytes), viability and three cytokines commonly associated with CRS (TNF-a, IL-6 and IL-10). Cellular phenotypes are then identified using a clustering algorithm and cytokine release level (response) is estimated using Gaussian mixture modeling methods. The response of the antibody of interest is now compared to a training set comprised of two classes of commercially available therapeutics, one class containing compounds that are known to induce CRS in patients and the other class containing compounds that are known to not induce CRS. After comparison with the training set, probability of CRS with the antibody of interest is estimated. Such a system would provide a robust method to evaluate the potential for CRS with a therapeutic antibody and also identify the subpopulations mediating this process. doi:10.1016/j.cyto.2008.07.147
107 Bioactive chicken IL-12: Plant-derived protein production platform compatible with veterinary vaccine applications Giuliana Medrano 1, Nathan Stephans 2, David Radin 2, Maureen C. Dolan 1, Carole L. Cramer 1,2, 1 Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA, 2 Biostrategies LLC. Jonesboro, AR, USA
Abstracts / Cytokine 43 (2008) 243–262
promoter reveals several STAT binding sites, suggesting that ps20 expression may be regulated transcriptionally by IFN. Using different truncated ps20 promoter constructs, we present data to describe the effects of IFN on that transcriptional regulation of ps20.
sensorineural hearing loss associated with inflammatory mediators such as TNF-a commonly found in the middle ear cleft of otitis media patients is still unclear from this experiment.
doi:10.1016/j.cyto.2008.07.143
doi:10.1016/j.cyto.2008.07.145
103 Experimental assessment of photodynamic therapy with 5,10,15,20-tetrakis(methoxyphenyl)-porphyrins in rat walker tumor Adriana G. Filip 1, Simona V. Clichici 1, Adriana V. Muresan 1, Claudia Gherman 2, Doina Daicoviciu 1, Rodica M. Ion 3, Simina Dreve 4, Nicoleta Decea 1, Remus Moldovan 1, 1 Department of Physiology, ‘‘Iuliu Hatieganu” University of Medicine and Pharmacy ClujNapoca, Romania, 2 Surgery Department, Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania, 3 National R&D Institute of Chemistry and Petrochemistry—ICECHIM, Bucharest, Romania, 4 National R&D Institute of Isotopic and Molecular Technologies, Cluj-Napoca, Romania
105 Cytokine evaluation of seven different respiratory infections Matthew C. Groll, Peter Bellos, Quansys Biosciences, Logan, Utah USA. Positive samples from Influenza A, B, Parainfluenza 1,2,3
PDT-mediated oxidative stress elicits direct tumor cell damage as well as microvascular injury. In addition to this direct killing process, tumor eradication also arises from an acute inflammatory response featured by an increased level of various mediators in the PDT-treated tumor area (IL-1 b, G-CSF, IL- 8, and MIP-2). To improve this treatment new photosensitizes are being synthesized and tested. Our study evaluates the effects of PDT with 5,10,15,20-tetrakis- (methoxyphenyl)-porphyrins (TMPP) and zinc-5, 10,15,20-tetrakis-(methoxyphenyl)-porphyrins (Zn-TMPP) upon the serical and tumoral levels of ROS, IL4 and TNFa on Wistar male rats bearing Walker carcinosarcoma 256. Rats were randomly divided into groups as follows: group 1—no treatment, group 2—the tumors were only irradiated, group 3 were given TMPP 10 mg/kg b.w. and irradiated, group 4—were given Zn-TMPP 10 mg/kg b.w. and irradiated, group 5—were given 5-aminolevulinic acid (5-ALA) 250-mg/kg b.w. and irradiated. Laser light (at fluency rate 100 J/cm2 and pulse frequency rate 10 kHz) at 685 nm was delivered to tumors for 15 min following 24 h after drug administration. The MDA levels in serum at 24 h after treatment were lower than those obtained with 5-ALA (TMPP: 1.37 ± 0.13: ZnTMPP: 1.44 ± 0. 21 nmoles/mg protein vs. 3.37 ± 0.70 nmoles/mg protein, p < 0,01). Regarding the levels of carbonylated proteins PDT with TMPP and ZnTMPP showed significantly increased levels compared to the control group (TMPP: 1.08 ± 0.39; ZnTMPP: 1.03 ± 0.22 vs. control: 0.61 ± 0.07 nmoles/mg protein; p < 0,01). These results suggest that the oxygen reactive species are involved in PDT action. Effect of PDT with TMPP and ZnTMPP on the immune system involves enhanced cytokine secretion (IL4 and TNFa), which may account for the subsequent tumor eradication by PDT. doi:10.1016/j.cyto.2008.07.144
104 Role for tumor necrosis factor-alpha (TNF-a) in hearing loss and in aquaporin-4 (AQP-4) expression Min Kyo Jung 1, Song E. Kim 1, Sang W. Yeo 1, Steven K. Juhn 2, 1 Department of Otolaryngology-HNS, School of Medicine, The Catholic University of Korea, Seoul, Korea, 2 Department of Otolaryngology, University of Minnesota Medical School, Minneapolis, MN, USA Sensorineural hearing loss is rare, but one of the serious sequelae of otitis media. Although the mechanism has not yet been clarified, inflammatory process of middle ear has been known to be propagated to inner ear possibly through the round window membrane, and to bring about sensorineural hearing loss. After inflammatory mediators present or produced in the middle ear cleft such as endotoxin, lipopolysachharides, nitric oxide, reactive oxygen species, arachidonic acid metabolites and cytokines enter the inner ear, they can disturb inner ear homeostasis and function, and trigger ongoing inflammatory process there. The dysfunction of the inner ear, especially hearing loss, seems to be brought about by direct ototoxicity to the hair cells or by disturbances in homeostasis associated with potassium ion recycling. In addition to the role of gap junctions and ion pumps that actively transporting ions in the cochlea, aquaporins are known to be exist in the cochlea as a passive water channel. Among the subtypes of aquaporins exist in the cochlea, aquaporin 4 (AQP4) is the most abundant and concentrate in the supporting cells neighboring hair cells, and seem to have function to cope with osmotic gradient during the excitation of hair cells with influx of potassium ions. For AQP4 knock out mice have been reported to have hearing loss, AQP4 seems to be related to the hearing loss somehow when the inner ear homeostasis is disturbed. We demonstrated that TNF-a, a principal proinflammatory cytokine in the pathogenesis of otitis media, induced sensorineural hearing losses in rats experimentally. TNF receptor type I (TNFRI) proved to be expressed in the organ of Corti and lateral ligament in the rat cochlear. TNF-a may induce sensorineural hearing loss through the mechanisms such as induction of nitric oxide synthase and the disturbances in the physiologic potassium ion recycling in the cochlear. However, the role for AQP4 expression in the pathogenesis of
Adenovirus and respiratory syncytial virus (RSV) were tested for 16 different cytokines to evaluate the presence or absences of cytokines during acute infection. Two samples from each infection were taken and tested in duplicate using the Quansys Biosciences Human Cytokine Screen Plus Array. This panel contains 50 nl spots of capture antibodies to the following cytokines; IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-23, IFNc, TNFa and TNFb. The samples were prepared in HAMA treated buffer. The samples were then loaded into a 96 well plate and incubated for 1 hour at room temperature. The plate was then washed and incubated with detection antibody mix with antibodies for each of the 16 assays. Post incubation, the plate was washed again and incubated with streptavidin-HRP and incubated for 15 min. The plate was then washed and chemiluminescent substrate was added. The plate was imaged for 1 min using a Quansys Imager. The Flu A, Flu B and RSV samples showed a typical inflammatory response with a high expression of IL-6 and IL-8. The Para 1 samples showed both IL-6 and IL-8 responses with one sample showing high values for IL-1a, IL-1b, IL-5 and TNFa. Para 3 samples showed similar IL-1a, IL-6 and IL-8 elevated values. Yet, one sample showed high levels of IL-5, IL12p70 and IL-13. The Adenovirus samples showed similar cytokine expression levels, IL-1b, IL-6, IL-8, IL-10, IFNc and TNFa. Many of the cytokine responses were expected such as IL-1b, IL-6 and IL-8 as seen in typical inflammation conditions. However, it was interesting to observe the disparity between samples of the same disease conditions ranging with very different cytokine profiles such as was observed in the Parainfluenza 1, 2 and 3 samples. doi:10.1016/j.cyto.2008.07.146
106 Strategy for a robust preclinical human assay to assess potential for cytokine release syndrome with therapeutic antibodies Ram Achuthanandam, Peter J. Bugelski, George Treacy, Renold J. Capocasale, Toxicology and Investigative Pharmacology, Centocor R&D, Radnor, PA, USA Cytokine release reactions are associated with administration of some therapeutic antibodies. These reactions vary in clinical significance from mild fever and nausea to severe reactions including hypotension, tachycardia and multi-organ failure. When these symptoms are anticipated they can be managed by prophylactic treatment with a wide variety of compounds. Here we present a system where we can assess potential for cytokine release syndrome (CRS) when administering therapeutic antibodies using polychromatic flow cytometry and a pattern recognition methodology. The antibody of interest is presented to human whole blood using solid phase beads (24 h incubation). A transport inhibitor is added to block cytokine secretion from within the cells during the last phase of the incubation. After incubation, the cells are washed, fixed and stained for a 7-color panel interrogating cell surface phenotype (CD19: B-cell, CD2: T-cells and NK cells and CD15: monocytes and granulocytes), viability and three cytokines commonly associated with CRS (TNF-a, IL-6 and IL-10). Cellular phenotypes are then identified using a clustering algorithm and cytokine release level (response) is estimated using Gaussian mixture modeling methods. The response of the antibody of interest is now compared to a training set comprised of two classes of commercially available therapeutics, one class containing compounds that are known to induce CRS in patients and the other class containing compounds that are known to not induce CRS. After comparison with the training set, probability of CRS with the antibody of interest is estimated. Such a system would provide a robust method to evaluate the potential for CRS with a therapeutic antibody and also identify the subpopulations mediating this process. doi:10.1016/j.cyto.2008.07.147
107 Bioactive chicken IL-12: Plant-derived protein production platform compatible with veterinary vaccine applications Giuliana Medrano 1, Nathan Stephans 2, David Radin 2, Maureen C. Dolan 1, Carole L. Cramer 1,2, 1 Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA, 2 Biostrategies LLC. Jonesboro, AR, USA