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HCV-RNA and inflammasome is modulated by PEGylated interferon (PEG-IFNα-2b) monotherapy in chronic hepatitis c patients
Abstracts/Clinical Biochemistry 43 (2010) 775–790
Results: Hemolysis causes a decrease of haptoglobin with the emergence of a haptoglobin–hemoglobin complex on the cathodic side of the α2-macroglobulin. Free hemoglobin is observed between transferrin and complement after haptoglobin saturation (around 2 g/L of hemoglobin added). Intralipid and Omnipaque™ interferences are observed between the orosomucoid and α1-antitrypsin. C3d appeared in early gammaglobulin region after 72 h at 4 °C, and after 24 h at room temperature. Fibrinogen is substantially at the same position as C3d while CRP is beside C3d, on the cathodic side. Conclusions: CZE-HR SPE can detect and differentiate intravascular hemolysis from in vitro hemolysis, α1-globulin increase from lipid or radiocontrast agent interference and paraprotein from Cd3, fibrinogen and CRP. Adequate knowledge of interferences allows optimal use of this new clinical application. doi:10.1016/j.clinbiochem.2010.04.022
117 The antagonistic role of Gcn5p and Hda1p in the promoter of anaphase promoting complex regulators in Saccharomyces cerevisiae A.I. Islam, E.T. Turner, T.A.A.H. Harkness Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Canada Background: Histone acetyltransferases (HATs) and histone deacetylases (HDACs) play central roles in chromatin metabolism, yet their in vivo functions and regulation remain largely unknown. HDAC inhibitors, aberrant histone acetylation and DNA methylation are under investigation for their potential as therapeutic agents/diagnostic markers in cancer. HATs and HDACs are conserved from yeast to humans, thereby making yeast an attractive model system for clinically relevant research. Objective: To explore the genetic interactions of HATs and HDACs with anaphase promoting complex (APC) subunits and their subsequent effects on chromatin metabolism. Methods: The methods used were spot dilution, Western blotting, Northern blotting, rtPCR and chromatin immunoprecipitation (ChIP). Results: Deletion of either the GCN5 or HDA1 gene leads to a similar temperature sensitive (ts) phenotype of the apc5CA allele. However, deletion of both GCN5 and HDA1 nullify each other so that their ts effects disappear and go back to the apc5CA phenotype. Compared to WT, the mRNA levels were decreased 20–30% in Δapc5gcn5, increased 20–30% in Δapc5hda1 and set somewhere in between in Δapc5gcn5hda1. These transcript level variations of all APC regulators comply with their promoter H3(K9/K14) acetylation. The overexpression of GCN5-HA and HDA1-HA and their subsequent promoter binding was followed by ChIP analysis. The Gcn5-HA binding was increased 2–3 fold in the promoter in Δgcn5hda1, compared to Δgcn5, which indicates that Hda1p prevents the access of Gcn5p in the promoter of APC regulators. Conclusion: Collectively, the data indicate that Hda1p prevents the recruitment of Gcn5p in the promoter of APC regulators. doi:10.1016/j.clinbiochem.2010.04.023
118 HCV-RNA and inflammasome is modulated by PEGylated interferon (PEG-IFNα-2b) monotherapy in chronic hepatitis c patients G. Neuman Manuelaa,b, Gadi G. Katzb, Ankit Patela,b, M. Izabella Malkiewiczb, Rustan Esguerrab Hepatitis Interventional Therapy Group a University of Toronto, Toronto, ON, Canada b In Vitro Drug Safety and BioTechnology, Toronto, ON, Canada Objective: To evaluate the inflammasome in HCV patients by correlating serum apoptosome, interleukins, RANTES, pathogen-
781
associated-molecular-pattern (PAMP), plasminogen-activator-inhibitor 1(PAI-1), tumour necrosis factor-alpha (TNFα), and transforming growth factor-beta (TGFβ) levels to (1) the severity of HCV and (2) the responses to PEG-IFNα-2b. Methods: 180-non-cirrhotic patients were part of a randomized, double-blind clinical trial PegIntron™ (Schering-Plough) that studied the efficacy of 0.5, 1.0 and 1.5 mg/kg/week PEG-IFNα-2b delivered over 48 weeks. In each dosage group the patients were sub-stratified by viral response to the PEG IFN as follows: sustainedresponse-SR [HCV-RNA undetectable 6 months after therapy ended (ET)], relapse-response-RR (HCV-RNA undetectable ET) or noresponse-NR (detectable HCV-RNA at ET). Serum inflammasome markers were quantitatively measured by ELISA. Significance among the groups was determined by Students-t-test with Bonferonni correction. The χ2 test or Fisher's exact test compared frequencies between groups. Results: At entry, groups showed no statistical difference regarding demographics, viral load, genotype, apoptosome, inflammasome, histological-activity-index (HAI) and fibrosis score. HAI and Metavirfibrosis (MF) scores were distributed as follows: (HAI → 3-zero, 47mild (HAI 1), 121-moderate (HAI 2) and 9-high (HAI 3); fibrosis → 5MF0; 152-MF1; 13-MF2; 10-MF3). HAI and TNFα levels correlated closely across all patients (r = 0.92, p < 0.001). IL-8 and RANTES increased significantly at HAI-3 versus HAI-1-2. TGFβ increased significantly with the severity of fibrosis. TNFα and apoptosis were lower at the base-line in SR versus RR and NR. This study illustrates a correlation between HCV-RNA and HAI-reduction. Conclusion: Low baseline serum TNFα and apoptosome are predictors for SR. PEG-IFNα-2 b reduces inflammasome contributing to reduced fibrosis. doi:10.1016/j.clinbiochem.2010.04.024
119 Early changes in biomarkers determined by LC/MS/MS and ELISA based methods in a phase I/II study assessing an anti-metastatic agent P. Kavsak, M. Henderson, H. Hirte, S. Hotte McMaster University, Hamilton, ON, Canada Objective: In cancer patients with advanced metastatic disease using a mass spectrometry (MS)-iTRAQ approach we identified macrophage stimulating protein (MSP) and l-selectin being up-regulated (≥1.5 fold from baseline/day1) in a subset of non-responders early after treatment with an anti-metastatic agent. The aim of the present study was to determine whether an antibody based methodology (ELISA or biochip) could confirm these observations. Methods: The study group (n = 8) consisted of subjects with cancers expressing the CXCR4 receptor with metastatic disease that were non-responsive to current therapy. Treatment with an investigational anti-metastatic drug (CTCE-9908) over 28 days identified 2 responders (breast; colorectal cancers) and 6 nonresponders (melanoma; colorectal; 4 breast cancers) by RECIST criteria. EDTA plasma (stored − 80 °C) collected on days 1, 5, and 19 of treatment were thawed and assayed for l-selectin by biochip array (Clin Biochem 2009; 42:1162–5) and for MSP by ELISA (R&D systems). Results: In the subset of non-responders (n = 3) the average fold change for MSP (iTRAQ ratios:day5/day1) as determined by MS was 1.70 (SD = 0.11) compared to 0.96 (SD = 0.19) as determined by ELISA (p = 0.019, t-test). Overall, there was no difference in the average fold changes as determined by ELISA in the 6 non-responders versus the 2 responders at both days 5 and 19 (1.21 vs. 1.19; p = 0.47). The average fold changes at both time points for l-selectin (biochip array) was
Results: Hemolysis causes a decrease of haptoglobin with the emergence of a haptoglobin–hemoglobin complex on the cathodic side of the α2-macroglobulin. Free hemoglobin is observed between transferrin and complement after haptoglobin saturation (around 2 g/L of hemoglobin added). Intralipid and Omnipaque™ interferences are observed between the orosomucoid and α1-antitrypsin. C3d appeared in early gammaglobulin region after 72 h at 4 °C, and after 24 h at room temperature. Fibrinogen is substantially at the same position as C3d while CRP is beside C3d, on the cathodic side. Conclusions: CZE-HR SPE can detect and differentiate intravascular hemolysis from in vitro hemolysis, α1-globulin increase from lipid or radiocontrast agent interference and paraprotein from Cd3, fibrinogen and CRP. Adequate knowledge of interferences allows optimal use of this new clinical application. doi:10.1016/j.clinbiochem.2010.04.022
117 The antagonistic role of Gcn5p and Hda1p in the promoter of anaphase promoting complex regulators in Saccharomyces cerevisiae A.I. Islam, E.T. Turner, T.A.A.H. Harkness Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Canada Background: Histone acetyltransferases (HATs) and histone deacetylases (HDACs) play central roles in chromatin metabolism, yet their in vivo functions and regulation remain largely unknown. HDAC inhibitors, aberrant histone acetylation and DNA methylation are under investigation for their potential as therapeutic agents/diagnostic markers in cancer. HATs and HDACs are conserved from yeast to humans, thereby making yeast an attractive model system for clinically relevant research. Objective: To explore the genetic interactions of HATs and HDACs with anaphase promoting complex (APC) subunits and their subsequent effects on chromatin metabolism. Methods: The methods used were spot dilution, Western blotting, Northern blotting, rtPCR and chromatin immunoprecipitation (ChIP). Results: Deletion of either the GCN5 or HDA1 gene leads to a similar temperature sensitive (ts) phenotype of the apc5CA allele. However, deletion of both GCN5 and HDA1 nullify each other so that their ts effects disappear and go back to the apc5CA phenotype. Compared to WT, the mRNA levels were decreased 20–30% in Δapc5gcn5, increased 20–30% in Δapc5hda1 and set somewhere in between in Δapc5gcn5hda1. These transcript level variations of all APC regulators comply with their promoter H3(K9/K14) acetylation. The overexpression of GCN5-HA and HDA1-HA and their subsequent promoter binding was followed by ChIP analysis. The Gcn5-HA binding was increased 2–3 fold in the promoter in Δgcn5hda1, compared to Δgcn5, which indicates that Hda1p prevents the access of Gcn5p in the promoter of APC regulators. Conclusion: Collectively, the data indicate that Hda1p prevents the recruitment of Gcn5p in the promoter of APC regulators. doi:10.1016/j.clinbiochem.2010.04.023
118 HCV-RNA and inflammasome is modulated by PEGylated interferon (PEG-IFNα-2b) monotherapy in chronic hepatitis c patients G. Neuman Manuelaa,b, Gadi G. Katzb, Ankit Patela,b, M. Izabella Malkiewiczb, Rustan Esguerrab Hepatitis Interventional Therapy Group a University of Toronto, Toronto, ON, Canada b In Vitro Drug Safety and BioTechnology, Toronto, ON, Canada Objective: To evaluate the inflammasome in HCV patients by correlating serum apoptosome, interleukins, RANTES, pathogen-
781
associated-molecular-pattern (PAMP), plasminogen-activator-inhibitor 1(PAI-1), tumour necrosis factor-alpha (TNFα), and transforming growth factor-beta (TGFβ) levels to (1) the severity of HCV and (2) the responses to PEG-IFNα-2b. Methods: 180-non-cirrhotic patients were part of a randomized, double-blind clinical trial PegIntron™ (Schering-Plough) that studied the efficacy of 0.5, 1.0 and 1.5 mg/kg/week PEG-IFNα-2b delivered over 48 weeks. In each dosage group the patients were sub-stratified by viral response to the PEG IFN as follows: sustainedresponse-SR [HCV-RNA undetectable 6 months after therapy ended (ET)], relapse-response-RR (HCV-RNA undetectable ET) or noresponse-NR (detectable HCV-RNA at ET). Serum inflammasome markers were quantitatively measured by ELISA. Significance among the groups was determined by Students-t-test with Bonferonni correction. The χ2 test or Fisher's exact test compared frequencies between groups. Results: At entry, groups showed no statistical difference regarding demographics, viral load, genotype, apoptosome, inflammasome, histological-activity-index (HAI) and fibrosis score. HAI and Metavirfibrosis (MF) scores were distributed as follows: (HAI → 3-zero, 47mild (HAI 1), 121-moderate (HAI 2) and 9-high (HAI 3); fibrosis → 5MF0; 152-MF1; 13-MF2; 10-MF3). HAI and TNFα levels correlated closely across all patients (r = 0.92, p < 0.001). IL-8 and RANTES increased significantly at HAI-3 versus HAI-1-2. TGFβ increased significantly with the severity of fibrosis. TNFα and apoptosis were lower at the base-line in SR versus RR and NR. This study illustrates a correlation between HCV-RNA and HAI-reduction. Conclusion: Low baseline serum TNFα and apoptosome are predictors for SR. PEG-IFNα-2 b reduces inflammasome contributing to reduced fibrosis. doi:10.1016/j.clinbiochem.2010.04.024
119 Early changes in biomarkers determined by LC/MS/MS and ELISA based methods in a phase I/II study assessing an anti-metastatic agent P. Kavsak, M. Henderson, H. Hirte, S. Hotte McMaster University, Hamilton, ON, Canada Objective: In cancer patients with advanced metastatic disease using a mass spectrometry (MS)-iTRAQ approach we identified macrophage stimulating protein (MSP) and l-selectin being up-regulated (≥1.5 fold from baseline/day1) in a subset of non-responders early after treatment with an anti-metastatic agent. The aim of the present study was to determine whether an antibody based methodology (ELISA or biochip) could confirm these observations. Methods: The study group (n = 8) consisted of subjects with cancers expressing the CXCR4 receptor with metastatic disease that were non-responsive to current therapy. Treatment with an investigational anti-metastatic drug (CTCE-9908) over 28 days identified 2 responders (breast; colorectal cancers) and 6 nonresponders (melanoma; colorectal; 4 breast cancers) by RECIST criteria. EDTA plasma (stored − 80 °C) collected on days 1, 5, and 19 of treatment were thawed and assayed for l-selectin by biochip array (Clin Biochem 2009; 42:1162–5) and for MSP by ELISA (R&D systems). Results: In the subset of non-responders (n = 3) the average fold change for MSP (iTRAQ ratios:day5/day1) as determined by MS was 1.70 (SD = 0.11) compared to 0.96 (SD = 0.19) as determined by ELISA (p = 0.019, t-test). Overall, there was no difference in the average fold changes as determined by ELISA in the 6 non-responders versus the 2 responders at both days 5 and 19 (1.21 vs. 1.19; p = 0.47). The average fold changes at both time points for l-selectin (biochip array) was