Gene Expression Profiling of Alcoholic Liver Disease in the Baboon (Papio hamadryas) and Human Liver
2003; Elsevier BV; Volume: 163; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63587-0
ISSN1525-2191
AutoresDevanshi Seth, Maria A. Leo, Peter H. McGuinness, Charles S. Lieber, Yvonne Brennan, Rohan B. H. Williams, Xin Wang, Geoffrey W. McCaughan, Mark D. Gorrell, Paul Haber,
Tópico(s)Hepatitis B Virus Studies
ResumoThe molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD. The molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD. Alcoholic liver disease (ALD) remains a major cause of mortality and morbidities that include gastrointestinal hemorrhage, liver failure, hepatocellular carcinoma, and the need for liver transplantation. Alcohol has been identified as a direct hepatotoxin and various pathways of pathogenesis have been implicated but their respective contribution to liver injury in humans is not clear.1Hill DB Deaciuc IV Nanji AA McClain CJ Mechanisms of hepatic injury in alcoholic liver disease.in: Gitlin N McCullough AJ Clinics in Liver Disease. Saunders, Philadelphia1998: 703-721Google Scholar Presently, there is no satisfactory treatment for this disease. Recent studies, mostly in animal models, suggest that alcohol initiates liver injury via endotoxin,2Thurman II, RG Alcoholic liver injury involves activation of Kupffer cells by endotoxin.Am J Physiol. 1998; 275: G605-G611PubMed Google Scholar oxidative stress,3Fernandez-Checa JC Kaplowitz N Garcia-Ruiz C Colell A Mitochondrial glutathione: importance and transport.Semin Liver Dis. 1998; 18: 389-401Crossref PubMed Scopus (191) Google Scholar and inflammation.1Hill DB Deaciuc IV Nanji AA McClain CJ Mechanisms of hepatic injury in alcoholic liver disease.in: Gitlin N McCullough AJ Clinics in Liver Disease. Saunders, Philadelphia1998: 703-721Google Scholar Disease progression involves continuing liver injury, fibrosis, and impaired liver regeneration. It is likely that a more detailed understanding of the pathogenesis will lead ultimately to more effective strategies for prevention and treatment of liver injury. DNA array analysis has provided novel data regarding pathogenesis of human liver disease. Studies of gene expression profiling in humans with different forms of liver cirrhosis (autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, hepatitis C) from our laboratory have identified several novel genes and pathways in liver disease.4Shackel NA McGuinness PH Abbott CA Gorrell MD McCaughan GW Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.Gut. 2001; 49: 565-576Crossref PubMed Scopus (136) Google Scholar, 5Shackel NA McGuinness PH Abbott CA Gorrell MD McCaughan GW Insights into the pathobiology of hepatitis C virus-associated cirrhosis: analysis of intrahepatic differential gene expression.Am J Pathol. 2002; 160: 641-654Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar Accordingly, we propose that gene expression profiling will define prominently expressed pathways and new molecular targets in ALD. The severity of ALD may be increased by co-factors such as nutritional impairment and chronic viral infection. Accordingly, observations made in humans and attributed to the effects of alcohol may be a consequence of other pathological processes seen in alcoholics rather than alcohol itself or liver disease. It may be difficult to properly identify and control for these confounders. An important complementary approach for studies of human liver tissue is to determine the effects of alcohol in an appropriate animal model. Of the various animal models of ALD, the baboon model has been shown to reproduce sequential development of all of the liver lesions observed in human alcoholics without exposure to other hepatotoxins.6Lieber CS DeCarli L Rubin E Sequential production of fatty liver, hepatitis, and cirrhosis in sub-human primates fed ethanol with adequate diets.Proc Natl Acad Sci USA. 1975; 72: 437-441Crossref PubMed Scopus (165) Google Scholar For this study, the pathogenesis of ALD was studied in the baboon model by comparing intrahepatic gene expression of alcohol-fed and control baboons using cDNA array analysis. The studies were extended to human ALD by comparison of gene expression profiles in nondiseased and cirrhotic liver tissue. The arrays have identified similarities in the overall gene expression profile and differential expression of many genes including a number of annexins and related molecules. Animal experiments were performed in accordance with local ethical requirements. Animals were fed the Lieber-DeCarli diet, a nutritionally adequate liquid diet in which the control and ethanol animals are fed isocaloric diets in which dextrose is substituted for ethanol as described.6Lieber CS DeCarli L Rubin E Sequential production of fatty liver, hepatitis, and cirrhosis in sub-human primates fed ethanol with adequate diets.Proc Natl Acad Sci USA. 1975; 72: 437-441Crossref PubMed Scopus (165) Google Scholar Needle liver biopsies were obtained from alcohol-fed animals and relevant pair-fed controls (two biopsies from each animal). The histopathology of the biopsies showed cirrhosis, steatosis, and inflammation of the tissue as previously reported6Lieber CS DeCarli L Rubin E Sequential production of fatty liver, hepatitis, and cirrhosis in sub-human primates fed ethanol with adequate diets.Proc Natl Acad Sci USA. 1975; 72: 437-441Crossref PubMed Scopus (165) Google Scholar and illustrated in Figure 1. The biopsies were snap-frozen, and stored in liquid N2 until extraction of RNA was performed. Human liver samples from seven male patients with advanced ALD (end-stage cirrhosis without viral hepatitis or other defined liver disease; Table 1) and seven nondiseased (donor) livers with normal histopathology, were obtained at the time of liver transplantation according to procedures approved by the Central Sydney Area Health Service (Royal Prince Alfred Hospital Zone) Ethics Committee guidelines. Explant tissues were snap-frozen in liquid N2 and stored at −70°C until further use.Table 1Human Liver ALD Patient ProfilesPatientAgeHistopathology159Micronodular cirrhosis, mild steatosis, alpha-1 antitrypsin deficiency, moderate bile duct proliferation, mild hepatic dysplasia257Micronodular cirrhosis, mild steatosis, focal Mallory's hyaline, moderate chronic inflammation in fibrous septa333Micronodular cirrhosis, diffuse steatosis, bile duct proliferation, scattered Mallory's hyaline, florid ductular proliferation, marked parenchymal iron deposition460Micronodular cirrhosis, minimal steatosis, chronic inflammation in fibrous septa, florid bile duct proliferation, focal piece-meal necrosis, focal hepatocyte dysplasia551Micro- and macro-nodular cirrhosis, increased hepatocyte iron staining, mild intracanalicular cholestasis, prominent bile duct proliferation652Micronodular cirrhosis, bile ductular proliferation, abundant Mallory's hyaline, mild parenchymal iron752Micronodular cirrhosis, prominent bile ductular proliferation, focal piece-meal necrosis Open table in a new tab Total RNA from biopsies was isolated in the presence of glycogen (250 μg/ml) using Trizol (Life Technologies, Inc., Gaithersburg, MD) as described.7Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar The RNA quality was verified by agarose gel electrophoresis and ensuring that the absorbance ratio (A260/280) was 1.8 to 2.1. The amount of RNA from baboon liver biopsies (50 to 80 μg) was insufficient for replicate gene array analysis. Therefore, RNA was amplified using the SMART-LD (switch mechanism at the 5′ end of reverse transcript-long distance) PCR (long distance PCR)-based exponential amplification system (Clontech, Palo Alto, CA) as described.7Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar SMART-LD PCR products have been validated for maintenance of gene expression levels after amplification with real-time RT-PCR.7Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar The purified samples could be stored for up to 4 weeks at −20°C. Amplified DNA (500 ng for each ethanol-fed and control baboon) was labeled with α-32P-dATP (10 μCi/μl, 3000 Ci/mmol; NEN Life Sciences Inc., Boston, MA) and Klenow fragment (MBI Fermentas GMBH, St. Leon-Pot, Germany). The probe was enriched for cDNAs spotted on the Atlas human 3.6K arrays using cDNA synthesis (CDS) primer (CDS 1.2I, CDS 1.2II, and CDS 1.2III provided in the Clontech kit) in the same reaction mix. Purified probes at ∼106 cpm/ml were hybridized to the arrays at 65°C overnight. Membranes were washed and exposed to phosphor imaging (Cyclone Phosphor Imager; Packard Instrument Company, Meriden, CT) for 24 to 96 hours.7Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar The experiment was performed four times with the same or a different batch of SMART-generated samples. DNA-membrane array images were analyzed with AtlasImage (version 2.1, Clontech) software. Further analysis was performed using nonlinear regression of raw gene expression dpm values for control versus experimental samples for each membrane. The predicted value for each gene was calculated from the regression equation and an expression ratio was calculated as an observed:predicted ratio for each gene.4Shackel NA McGuinness PH Abbott CA Gorrell MD McCaughan GW Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.Gut. 2001; 49: 565-576Crossref PubMed Scopus (136) Google Scholar, 7Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar Genes with expression ratios of 1.5 or more in at least three of the four replicate DNA array experiments and >1.0 in all four experiments were considered to be up-regulated. RNA was extracted with a RNAqueous-Midi kit (Ambion, Austin, TX) following the manufacturer's instructions (RNAqueous-Midi Instruction Manual). Explant liver tissue (0.15 × g per sample) and nondiseased liver tissue from wedge biopsies were individually homogenized in the guanidine lysis buffer provided in the kit using an Ultra-Turrax T8 tissue homogenizer (IKA Labortechnik, Staufen, Germany). The homogenized tissue was applied to the glass fiber filter (included in the kit), washed extensively with the buffer before eluting RNA in water. The quality of RNA was tested as above and all samples were stored at −70°C. Total RNA from the ALD and nondiseased explants were pooled in each group in equal amounts. Pooled RNA (60 μg) from ALD and nondiseased group was directly labeled with either dUTP-Cy5 or dUTP-Cy3 fluorophores, respectively, and 4 μg Oligo-dT (Roche, Basil, Switzerland) using SuperscriptII (Invitrogen Corporation, Rockville, MD) at the time of reverse transcription. Five μl of Lucidea Microarray ScoreCard Spike Mix (Amersham Pharmacia Biotech) was also added to the RNA mix. The RNA mix was denatured at 65°C for 10 minutes and added to 5× first strand buffer (Invitrogen); 400 U SuperScript II; 0.5 mmol/L dATP, dTTP, dGTP; 0.3 mmol/L dCTP (Amersham Pharmacia Biotech); 11 mmol/L DTT; and 16 U RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen). Fluorescence-labeled d-UTPs [Cy3 (PA53022) or Cy5 (PA55022) (2.5 nmol), Amersham Pharmacia Biotech)] were added and the reaction incubated at 42°C for 2 hours. An additional 200 U SuperScript II was added to the tube after 1 hour of incubation. Adding 15 μl of fresh 0.1 mol/L NaOH and incubating at 70°C for 10 minutes terminated the reaction. The cDNA mixture was neutralized by adding 15 μl of 0.1 mol/L HCl. The labeled cDNA was purified with a Qia-Quick PCR purification kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The probe was concentrated using a Microcon YM-30 filter (Millipore, Bedford, MA). Briefly, the two RNA reactions (Cy3 and Cy5) were combined and added to the microcon filter. TE buffer (400 μl) containing 20 μg each of Cot1 human DNA (Invitrogen), poly A RNA (Sigma, St. Louis, MO) and tRNA (Invitrogen) was added to the filter. The probe was concentrated to a final volume of 20 μl. Probe was denatured in 4.25 μl of 20× standard saline citrate (SSC) and 0.65 μl of 10% sodium dodecyl sulfate for 2 minutes at 99°C. The human 6K cDNA glass microarray (RGHu6K on Telechem SuperAmine slides from The Clive and Vera Ramaciotti Centre for Gene Function Analysis, University of New South Wales, Kensington, Australia) was hybridized with 24.5 μl of the probe mixture in four replicate experiments. The slide was placed in a 50-ml Falcon humidified using 2× SSC and incubated in the dark at 65°C overnight in a Mini Hybridization Oven (ThermoHybaid, Ulm, Germany). The slide was washed once in 2× SSC, 0.1% sodium dodecyl sulfate for 1 minute to remove the coverslip, once in 1× SSC for 30 seconds, and once in 0.2× SSC for 1 minute. All washing steps were performed at room temperature with gentle rocking. The slides were dried immediately by spinning in a tube at 600 rpm for 3 minutes. The slides were scanned with a GenePix 4000 microarray scanner and images and data were acquired with GenePix Pro 3.0 microarray image acquisition and image analysis software (Axon Instruments, Inc., CA). The scanner was set at 100% laser power with pixel resolution at 10 μm. The photomultiplier tube voltage varied depending on the signal intensity and was adjusted so that the total red intensity of each slide was equal to the total green intensity of that slide. Corrected intensities were calculated for both the red and green channels by subtracting the median measure of background pixel intensities from the median measure of spot pixel intensity for each spot as follows:IRcorr(corrected red intensity) = IRspot −I RbackgroundIGcorr(corrected green intensity) = IGspot−I Gbackground Data normalization was performed using the R statistical computing environment8Ihaka R Gentleman RR "R": a language for data analysis and graphics.J Comput Graph Statist. 1996; 5: 299-314Google Scholar using the statistical microarray analysis library.9Yang YH, Dudoit S, Luu P, Speed TP: Normalization for cDNA microarray data. Edited by Bittner ML, Chen Y, Dorsal AN, Dougherty ER. Microarrays: Optical Technologies and Informatics. Proc SPIE 2001: pp 141-152 (The International Society for Optical Engineering)Google Scholar This procedure is specific for normalization of data generated from two-dye experiments. Log ratio (M) and log average intensity (A) values were calculated for each spot as follows:M=LOG2(IRcorr÷ IGcorr)A=LOG22(IRcorr × IGcorr)Nonparametric, locally weighted regression curves (Lowess function with the f value set at 0.1) were fitted to the data points. Normalization using this technique adjusts the local mean of the log expression ratio to M = 0. Print-tip group Lowess normalization9Yang YH, Dudoit S, Luu P, Speed TP: Normalization for cDNA microarray data. Edited by Bittner ML, Chen Y, Dorsal AN, Dougherty ER. Microarrays: Optical Technologies and Informatics. Proc SPIE 2001: pp 141-152 (The International Society for Optical Engineering)Google Scholar, 10Yang YH Dudoit S Luu P Lin DM Peng V Ngai J Speed TP Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.Nucleic Acids Res. 2002; 30: 1-10Crossref PubMed Scopus (39) Google Scholar was chosen and Lowess curves were fitted to MA data from each print-tip group (ie, each block) separately. Using this normalization method the intensities of the spots within a particular print-tip group are normalized independently of the spots in other print-tip groups. Print-tip group Lowess normalization was chosen because it deals with the intraarray variability and print-tip effects by treating and comparing each block of the array separately, as opposed to global Lowess normalization. The low-intensity spots were eliminated using the upper 95th percentile of A values of water (negative control) spots on the array as the cut-off threshold. Intensities of gene spots comparable to the intensity of the water spots were considered to represent background signal and were therefore eliminated. Separate cut-off values were determined for each of the four replicate arrays. After normalization and elimination of low-intensity spots, duplicate spots for each gene were averaged. A gene was determined as being differentially expressed if its M value was greater than or equal to 1 or less than or equal to −1, which corresponds to a twofold differential expression ratio, in at least three of four arrays. Genes determined as being differentially expressed were annotated with its SwissProt description, function, and keywords using Database Referencing of Array Genes Online,11Bouton CM Pevsner J DRAGON View: information visualization for annotated microarray data.Bioinformatics. 2002; 18: 323-324Crossref PubMed Scopus (27) Google Scholar at: http://pevsnerlab.kennedykrieger.org/dragon.htm. Each differentially expressed gene was annotated. Where feasible, functional categories were allocated as they relate to liver biology from the literature. Two sets of cDNAs were synthesized for each RNA sample to serve as replicate. One μg of total RNA was subjected to first-strand cDNA synthesis using Superscript II (Life Technologies) and oligo dT (Roche). The cDNAs were diluted fivefold, stored in small aliquots at −70°C and 1 μl was used for real-time PCR. Real-time RT-PCR was performed and analyzed as described previously.7Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar, 12Yin JL Shackel NA Zekry A McGuinness PH Richards C Putten KV McCaughan GW Eris JM Bishop GA Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.Immunol Cell Biol. 2001; 79: 213-221Crossref PubMed Scopus (264) Google Scholar Primers for real-time RT-PCR were obtained from previous reports for: frizzled related protein, ubiquitin,13Shackel NA Gorrell MD McCaughan GW Gene array analysis and the liver.Hepatology. 2002; 36: 1313-1325Crossref PubMed Google Scholar glial cell-derived neurotrophic factor, neuromodulin (GAP43),13Shackel NA Gorrell MD McCaughan GW Gene array analysis and the liver.Hepatology. 2002; 36: 1313-1325Crossref PubMed Google Scholar annexin A2 light chain (p11),14Chetcuti A Margan SH Russell P Mann S Millar DS Clark SJ Rogers J Handelsman DJ Dong Q Loss of annexin II heavy and light chains in prostate cancer and its precursors.Cancer Res. 2001; 61: 6331-6334PubMed Google Scholar and cytochrome P450 (CYP) IIE1 (CYP2E1).7Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar Other primer sequences are defined below. Annexin A2 (heavy chain p36) forward: 5′ TCTGAGCTAAAAGCTTCCATGAA 3′, reverse: 5′ GGAGTGAAGAGGAAAGGA 3′; annexin A1 (ANXA 1) forward: 5′ CGATCTGAGGACTTTGGTGTGAAT 3′, reverse: 5′ GTTTGCTTGTGGCGCACTT 3′; annexin A7 (ANXA7) forward: 5′ GCTGCCAACTTCGATGCTATAAGA, reverse: 5′ CTGGTTCTCATCACGATTTCCC 3′; aldehyde dehydrogenase 1 forward: 5′ ATGCCGACTTGGACAATGC 3′, reverse: 5′ CCCCTTCTTTCTTCCCACTCTC 3′; interferon γ-induced protein-10 (CXCL10) forward: 5′ TGCTGCCTTATCTTTCT 3′, reverse: 5′ GAAAGCAGTTAGCAAGG 3′. The PCR products were sequence verified. All real-time RT-PCR studies were performed using duplicate cDNAs from individual samples (n = 8 for human and n = 4 for baboon). The experiment was performed at least twice for each set of cDNA and the number of transcript molecules per μg of RNA was calculated from DNA standards and normalized on ubiquitin data. The data are represented as means ± SD. Frozen human liver specimens were sectioned at 5 to 10 μm thickness at −17°C, collected onto gelatin/chrome alum slides, and fixed in 100% ethanol (BDH AnalaR, Kilsyth, Australia) for 15 minutes. Slides from −20°C were prewarmed at 37°C for 15 minutes before blocking, staining, and imaging as described previously15Levy MT McCaughan GW Abbott CA Park JE Cunningham AM Mueller E Retting WJ Gorrell MD Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis.Hepatology. 1999; 29: 1768-1778Crossref PubMed Scopus (262) Google Scholar, 16Ajami K Abbott CA Obradovic M Gysbers V Kähne T McCaughan GW Gorrell MD Structural requirements for catalysis, expression, and dimerization in the CD26/DPIV gene family.Biochemistry. 2003; 42: 694-701Crossref PubMed Scopus (63) Google Scholar using the reagents listed in Table 2. Goat anti-rabbit IgG-Alexa Fluor 594 and goat anti-mouse IgG-Alexa Fluor 488 (Molecular Probes, Eugene, OR) secondary antibodies (1:400 dilution) were used for detection.Table 2Primary AntibodiesAntigenClone/nameIsotypeSupplierWorking dilutionAnnexin II (H-50)Rabbit IgGSanta Cruz, Santa Cruz, CA5.0 μg/mlAnnexin IIZO14Mouse IgG1Zymed, South San Francisco, CA1.7 μg/mlCD34My10Mouse IgG1Becton Dickinson (Franklin Lakes, NJ)1:10Cytokeratin 7 (CK7)OV-TL 12/30Mouse IgG1DAKO (A/S, Glostrup Denmark)3.5 μg/mlAnti-human c-Kit (CD117)YB5.B8Mouse IgG1Pharmingen, Glostrup, CA1.7 μg/mlAnti-human FAPF19Mouse IgGW. Rettig; Levy et. al., 19991:5CD14UCH-M1Mouse IgG2aSanta Cruz2.0 μg/mlMacrophage CD68EBM11Mouse IgG1DAKO2.3 μg/mlT cell CD3UCHT 1Mouse IgG1DAKO3 μg/mlGFAPRabbit IgGDAKO1.0 mg/mlCD19HD37Mouse IgG1DAKO1.7 μg/mlSynaptophysinRabbit IgGNovocastra, Newcastle upon Tyne, UK1:40Mouse IgG1, controlMOPC-21Mouse IgG1Sigma, St. Louis, MO10 μg/mlImmunoglobulin Open table in a new tab Probes generated from SMART-amplified biopsy samples from control-fed and ethanol-fed baboons were successfully hybridized to Atlas human 3.6K cDNA arrays (array, Figure 2). Linear regression of raw gene expression values (average of four experiments in the three membranes of the 3.6K set: 1.2I R2 = 0.929, 1.2II R2 = 0.889, 1.2III R2 = 0.861) for control versus ethanol-treated samples revealed that of the 36% of genes detected twofold greater than background, 26% were differentially expressed (288 genes up-regulated and 43 genes down-regulated). Differential expression of CYP2E17Seth D Gorrell MD McGuinness PH Leo MA Lieber CS McCaughan GW Haber PS SMART amplification maintains representation of relative gene expression: quantitative validation by real time PCR and application to studies of alcoholic liver disease in primates.J Biochem Biophys Methods. 2003; 55: 53-66Crossref PubMed Scopus (56) Google Scholar and other genes is evident in Figure 2. Differential expression of genes in human ALD was evident as shown in the raw array image (Figure 3). Normalization of array data using print-tip group Lowess minimized the ratio bias generated from variable intensities across individual slides (Figure 4). Of the 57.5% detected genes, 8% (206 genes up-regulated and 76 genes down-regulated) were found to be differentially expressed after normalization and further analysis.Figure 4.Print-tip group Lowess function reduces intraslide intensity-dependent ratio variability. Box-whisker plots of a typical array without normalization (A), and print-tip group global Lowess normalization.9Yang YH, Dudoit S, Luu P, Speed TP: Normalization for cDNA microarray data. Edited by Bittner ML, Chen Y, Dorsal AN, Dougherty ER. Microarrays: Optical Technologies and Informatics. Proc SPIE 2001: pp 141-152 (The International Society for Optical Engineering)Google Scholar, 10Yang YH Dudoit S Luu P Lin DM Peng V Ngai J Speed TP Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.Nucleic Acids Res. 2002; 30: 1-10Crossref PubMed Scopus (39) Google Scholar (B). M is defined in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The differentially expressed genes were assigned a broad category based on their biological functions or cellular location. A comparative global hepatic gene expression profile was similar in the baboon and human (Figure 5). A high proportion of genes, from both baboons and humans, were found in the following categories: fibrosis/extracellular matrix (ECM), metabolism, immune system, transcription, and cell signaling. Many genes appeared in the fibrosis/ECM category in both the baboon (19%) and human (24%) arrays. A comparatively large proportion of genes belong to the immune system (14%) or nuclear/transcription (12%) category in advanced human ALD, whereas the trafficking (9%), signaling (8%), and neural (6%) categories were more prominent in the baboon. The proportion of genes in the metabolism category was nearly double in the baboons (24%) as in the humans (12%). The complete lists of differentially expressed genes from the baboon and human arrays with Unigene and GenBank numbers is provided as supplementary material for placement on the journal web site. (These supplemental data are currently available on the following nonlinked web site: http://www.centenary.usyd.edu.au/pubs/additions/ALD/data.html). Selected differentially expressed genes from the most prominent categories with respect to disease pathology (fibrosis, energy metabolism, oxidant stress, immune related, trafficking, and neural), are listed for baboons (Table 3) and humans (Table 4). A number of the
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