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Originally posted on The Sports Headquarters | Last updated 1/2/13
In the midst of finishing up my first entry for The Football Standard my Twitter, shameless plug @sethcoxFB, blew up with news of a reported agreement being worked out between the Arizona Cardinals, my personal favorite football team, and Andy Reid, former head coach of the Philadelphia Eagles. While I was suspect at first about Reid and the Cardinals working on an agreement, the news of Reid and the Cardinals came out hours prior to any report of the Cardinals interviewing their own defensive coordinator Ray Horton in order to fulfill the Rooney Rule requirements, news came out later that indeed the Cardinals were in the process of interviewing Horton during the outbreak of the Reid to Arizona rumors. (Adam Schefter has now backtracked on his sources saying things are close in Arizona.) I don't want to speculate what's going to happen, that's for journalists to do, I want to look at the pros and cons of Andy Reid bringing his rotund presence to the desert and what my beloved #Birdgang (hat tip to @azbirdgangMC and @zoopaloop) may be able to expect in 2013 and beyond. The Good: The offense is likely to become a consistent top tier offense. That's a simplistic explanation in verbiage but will take on a distinctly different look from an aesthetics point of view, one that is surely to please fans. Since Reid took over the Eagles in 1999, running his own brand of the West Coast Offense, something we will surely get into if and when Reid is officially hired by the Cardinals, has put up points. In 14 seasons in Philadelphia, Reid's offense finished in the top 10 in points per game 8 times, even though he has employed 3 different offensive coordinators through that time, he was able to get there with each of the different OC's in tow. Add into that he has finished in the top 10 seven different times for yards per game, including a top 5 rushing offense in two of the last three seasons, and you know that his offenses will consistently produce and put up numbers, something that has been only present in the desert during the Kurt Warner era in Arizona. The Bad: Reid is widely regarded as a QB whisperer of sorts in the league. He was able to transform the careers of A.J Feeley before quickly dealing him for a second round pick to the Miami Dolphins and Kevin Kolb, who we Arizona fans know far too much about. Why is this bad? Well, outside of Donovan McNabb, Reid has never fully sustained QB success. What he did with McNabb is one of the more overlooked successes in NFL history, but he never won a Super Bowl with McNabb and fell short in numerous big games leading to the duo being overlooked in the aspect of great quarterback, head coach combos in history. That's not the bad, the bad is after. While Kolb had mild success in limited time in Philadelphia before being dealt, the idea was that Mike Vick would be the long term answer to Reid's search for McNabb's successor. This was one of the bigger flops in NFL history. Vick put on a historic display in 2010, and got significantly worse the following two seasons and is likely on his way out of Philadelphia along with Reid. This leads to the question: Was Reid successful because of McNabb or was McNabb successful because of Reid? We have seen the list of quarterbacks to put up serviceable numbers in a Reid lead offense, but we have only seen one that has had any semblance of elongated success, and the demise of Reid coincided greatly with McNabb's departure from Philadelphia. Can Reid squeeze the most out of Kevin Kolb's abilities, and how good are those abilities if Reid was so quick to jettison Kolb with the knowledge of Vick's injury history? Is Reid of the same ilk of Ken Whisenhunt in Arizona, a product of near Hall of Fame level quarterback play? These are questions that absolutely have to be asked before some Cardinals fans begin their glorification of Reid as a franchise savior. The Ugly: The biggest question coming into the Reid era in Arizona if this all comes to fruition: Can Andy Reid find a defensive coordinator? The Eagles consistently boasted one of the better defenses in the NFL in the early, mid and late 00′s with the late Jim Johnson calling the shots. In fact, from 2001-2004 the Eagles finished as the number two scoring defense three times in those four seasons. In fact the Eagles finished in the top ten in scoring defense in seven of Johnson's ten seasons as the defensive coordinator. After Johnson passed away, the defenses saw drops too: 19th, 21st, 10th, and 29th in 2012. It is no coincidence that the Eagles experienced their first one and done in the playoffs ever in 2009 and followed that up with a one and done in 2010. Reid may be an offensive savant, but he seems to be unable to find anyone to match wits with him on the defensive side of the ball. Is bringing in someone like Reid, a proven winner, a supposed quarterback and offensive genius worth risking losing a dynamic, and growing defensive coordinator like Ray Horton? I have gone back and forth on the idea of Horton as head coach, and while he isn't my first choice, I do favor him over a Reid because of the questions I have in the bad and ugly section. If it happens though, I am all in and will delete this post immediately, only for it to magically reappear once the Reid era comes to an end.
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Patent application title: BIOMARKERS FOR AUTOIMMUNE LIVER DISEASES AND USES THEREOF
Sergio Abrignani (Rapolano Terme (si), IT)
Mauro Bombaci (Milano (mi), IT)
Chiara Zingaretti (Ravenna (ra), IT)
IPC8 Class: AG01N3368FI
USPC Class:
506 9
Class name: Combinatorial chemistry technology: method, library, apparatus method of screening a library by measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Publication date: 2014-04-17
Patent application number: 20140106987
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The present invention relates to a method for diagnosis or prognosis of
liver autoimmune diseases by means of detecting specific biomarkers in
biological samples. The invention refers also to a method of monitoring
an autoimmune liver disease pathology status after treatment with surgery
and/or therapy in a subject with autoimmune liver disease, to kits and
microarrays to perform said methods.
1. An in vitro method of diagnosis or prognosis or evaluation of risk to
develop a liver autoimmune disorder belonging to the group of AIH and PBC
in a subject, comprising the steps of: a) contacting a biological sample
from the subject with a protein selected from the group consisting of: a
protein having the amino acid sequence SEQ ID No. 1, an allelic variant,
an orthologous, at least one immunological fragment and functional
equivalents thereof, under conditions appropriate for binding of
autoantibodies, if present in the biological sample, to said protein, and
b) detecting the presence of bound autoantibodies.
2. The method according to claim 1 wherein step a) is performed by contacting said biological sample with the protein as in claim 1 and at least one further protein selected from the group consisting of 16 proteins having amino acid sequence SEQ ID Nos. 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 22, 25, allelic variants, orthologous, immunological fragments and functional equivalents thereof.
3. The method according to claim 2 wherein step a) is performed by contacting said biological sample with three proteins having amino acid sequence SEQ ID Nos. 1, 11, 17, allelic variants, orthologous, immunological fragments or functional equivalents thereof.
4. The method according to claim 3 wherein step a) is performed by contacting said biological sample with four proteins having amino acid sequence SEQ ID Nos. 1, 10, 11, 17, allelic variants, orthologous, immunological fragments or functional equivalents thereof.
5. The method according to claim 4 wherein step a) is performed by contacting said biological sample with six proteins having amino acid sequence SEQ ID Nos. 1, 6, 8, 10, 11, 17, allelic variants, orthologous, immunological fragments or functional equivalents thereof.
6. The method according to claim 1 wherein the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, mucus, and fractions thereof.
7. The method of claim 1 wherein the biological sample is from an adult or from an adolescent.
8. The method according to claim 1 wherein the detection of said bound autoantibodies is performed by means of binding to specific ligands.
9. The method according to claim 8 wherein the ligands are conjugated with detecting means.
10. A method of monitoring an autoimmune liver disorder after treatment with surgery and/or therapy in a subject with said autoimmune liver disorder, comprising the step of following the modulation of proteins as claimed in claim 1.
11. The method of claim 1, wherein said proteins or functional equivalents thereof are displayed on one or more protein microarrays.
12. (canceled)
13. A solid support for an immunodiagnostic assay comprising a protein of claim 1 or immunological fragments or functional equivalents thereof.
14. An immunodiagnostic kit comprising the solid support of claim 13 and detecting means.
[0001] The present invention relates to the field of immunodiagnostic and/or prognostic of liver autoimmune diseases.
[0002] Autoimmune Liver Diseases (AILD) are chronic and progressive disorders with a poorly understood etiology. The most common AILD are Autoimmune Hepatitis (AIH) and Primary Biliary Cirrhosis (PBC).
[0003] Autoimmune Hepatitis (AIH) is a chronic necro-inflammatory disease and one of the most common autoimmune liver diseases AIH has an incidence of 1-2 per 100,000 per year, and a prevalence of 1-10/100,000. As with most of the other autoimmune diseases, it affects women more often than men (80%), with a sex ratio of about 3:1 (female to male) (Czaja A J. et al., 2010, Gastroenterology; Makol et al., 2011, Hepatitis research and treatment). Histologically it is characterized by: interface hepatitis and plasma cell infiltration; hypergammaglobulinemia is often present; a number of autoantibodies can be detected such as antinuclear antibodies (ANA), anti-Smooth Muscle Antibody (SMA), liver/kidney microsomal antibody (LKM-1), LC1, anti-actin, anti-ASGPR (Bogdanos D P. et al., 2009, Semin Liver Dis). Primary Biliary Cirrhosis (PBC) is a slowly progressing disease causing the destruction of small and medium-size intra-hepatic bile ducts (Selmi C. et al., 2011, Imm Cell Bio). It affects women in 90% of cases. The prevalence is estimated at 0.6-40/100.000. Ursodeoxicholic acid has been shown to improve serum biochemistry, histology and patient's survival (Muratori L. et al., 2010, Dig Liver Dis; Muratori L. et al., 2008, Clin Liver Dis). It is characterized by: anti-mitochondrial antibodies--AMA--(˜90%) and intrahepatic cholestasis (increased alkaline phosphatase--Alk Ph--, normal ultrasonographic--US--scan).
[0004] The detection of the AMA autoantibodies is performed routinely by immunofluorescence on fresh multi-organ sections (liver, kidney, stomach) from rodents, but this technique may present many intrinsic problems such as standardization and interpretation of the immuno-morphological patterns (Bogdanos et al., 2008, WJG). To overcome these methodological problems, the International Autoimmune Hepatitis Group established an internationally representative committee to define guidelines and develop procedures and reference standards for more reliable testing (Vergani et al., 2004, Journal of hepatology). In recent years, some AILD target-autoantigens have been identified and characterized (Zachou, K., et al., 2004; J of Autoimm Dis), but little is known on their pathogenetic role, and probably many autoantigens are still unknown. For autoantibodies to have a pathogenetic role, two features have to be met: (i) the autoantigen should be expressed on the target organ and exposed to autoantibodies, (ii) the autoantibodies should have functional activity. Song Q. et al. (2010, J. Proteome Res) described the identification of highly specific biomarkers and their validation for AIH. This study demonstrates that the combination of six autoantigens can be used to diagnose AIH-positive serum samples and that these autoantigens can be effectively used in protein microarray assays, as well as, in traditional ELISA-based assays.
[0005] US2009/0023162 discloses the methods for the identification of atypical antineutrophil cytoplasmic antibodies (ANCA), kits suitable for the same and application of said methods to the diagnosis of chronic inflammatory intestinal diseases and autoimmune liver diseases.
[0006] RU 2247387 (C1) provides a method involving determination of anti-mitochondrial antibodies, immunoglobulins such as IgA, IgM, IgG, gamma-globulins, anti-gliadin antibodies, and circulating immune complexes for the diagnosis of autoimmune liver injuries in patients with chronic hepatitis.
[0007] To date, however, there are no early and precise assays that can be used to identify individuals carrying or at risk of developing AILD. An early diagnosis is clearly important.
[0008] Dalekos G. 2002 European J. Int. Medicine, vol. 13, n. 5, pp. 293-303 and Jones D. E. 2000 Journ. Clin. Pathol. Vol. 53, n. 11, pp. 813-821 disclose some autoantigens in AIH and PBC.
[0009] Though the identification of some autoantibodies is within prior art documents, there is still the need to identify novel biomarkers, namely autoantibodies, to diagnose the liver autoimmune disease (AILD) and/or to discriminate between autoimmune and other liver pathologies, and/or to monitor the efficacy of patient treatments of liver autoimmune disease and the disease progression. In the present invention a subject with autoimmune liver disease is named "AILD or hepatic autoimmunity patient" and is affected by autoimmune liver disease, including AIH or PBC.
[0010] In the present invention, a panel of 17 autoantigens was identified in patients with AILD by protein array. In addition, 6 out of the 17 autoantigens were also validated by Dissociation Enhanced Lanthanide FluoroImmunoAssay method (DELFIA®), in patients diagnosed with liver autoimmune diseases, and showed individual sensitivities ranging from 42% to 74%. The combined assessment of these six autoantigens displays a 82%±4% sensitivity and almost 90%±3% specificity.
[0011] These six autoantigens represent novel markers of liver autoimmunity, such as AIH and PBC. These markers can display much higher sensitivity and specificity (Vs other diseases such as HCV and HBV) when compared to the benchmark markers (CYP2D6 & ASGPR, as described in the Methods section). Therefore, the autoantigens identified in the present invention are valuable tools for the development of a new serological assay that is easy to perform and is highly specific for AILD diseases. This assay could significantly contribute to an improvement of AILD diagnosis and to a discrimination between PBC and AIH.
[0012] It is therefore an object of the present invention an in vitro method of diagnosis or prognosis or evaluation of risk to develop a liver autoimmune disorder belonging to the group of AIH and PBC in a subject, comprising the steps of:
a) contacting a biological sample from the subject with a protein comprised in the group of: a protein having the amino acid sequence SEQ ID No. 1, an allelic variant, an orthologous, at least one immunological fragment or a functional equivalent thereof, under conditions appropriate for binding of autoantibodies, if present in the biological sample, to said protein, and b) detecting the presence of bound autoantibodies.
[0013] In the context of the instant invention the term "protein" includes:
[0014] i. the whole protein, allelic variants and orthologous thereof;
[0015] ii. any immunological, synthetic or recombinant or proteolytic fragment of a protein having the ability to be recognized by and bound to antibodies directed against the protein;
[0016] iii. any functional equivalent comprised in the group of synthetic or recombinant functional analogs having the ability to be recognized by and bound to antibodies directed against the protein.
[0017] In a preferred embodiment, step a) is performed by contacting said biological sample with the protein having the amino acid sequences SEQ ID No. 1 and at least one further protein selected from the group of 16 proteins having the amino acid sequences SEQ ID No. 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 22, 25, allelic variants, orthologous, immunological fragments or functional equivalents thereof.
[0018] In a further embodiment step a) is performed by contacting said biological sample with three proteins having the amino acid sequences SEQ ID No. 1, 11, 17 allelic variants, orthologous, immunological fragments or functional equivalents thereof.
[0019] In a further preferred embodiment step a) is performed by contacting said biological sample with four proteins having the amino acid sequences SEQ ID No 1, 10, 11, 17, allelic variants, orthologous, immunological fragments or functional equivalents thereof.
[0020] In a further preferred embodiment step a) is performed by contacting said biological sample with six proteins having the amino acid sequences SEQ ID No 1, 6, 8, 10, 11, 17 allelic variants, orthologous, immunological fragments or functional equivalents thereof.
[0021] Preferably the biological sample is comprised in the group of blood, serum, plasma, urine, saliva, mucus, or fractions thereof.
[0022] Preferably the biological sample is from an adult or from an adolescent.
[0023] Preferably the detection of said bound autoantibodies is performed by means of binding to specific ligands. More preferably the ligands are conjugated with detecting means.
[0024] It is another object of the invention a method of monitoring an autoimmune liver disorder after treatment with surgery and/or therapy in a subject with said autoimmune liver disorder, comprising the step of following the modulation of antibodies as disclosed.
[0025] In a preferred aspect said proteins or immunological fragments or functional equivalents thereof are displayed on one or more protein microarrays.
[0026] It is another object of the invention the use of a protein microarray comprising at least the proteins as defined or immunological fragments or functional equivalents thereof for performing the method according to the invention.
[0027] It is another object of the invention the use of a solid support for an immunodiagnostic assay comprising at least the proteins as defined or immunological fragments or functional equivalents thereof for performing the method according to the invention.
[0028] It is another object of the invention the use of an immunodiagnostic kit comprising the above solid support and detecting means for performing the method according to the invention.
[0029] In the present invention a subject with autoimmune liver disease is named "AILD or hepatic autoimmunity patient" and is affected by any autoimmune liver disease, as AIH or PBC.
[0030] HCV patients are patients diagnosed with hepatitis C and displaying autoreactive antibodies, or patients with HCV infection or patients with hepatitis C and non hepatic autoimmune diseases such as crioglobulinemia or thyroid dysfunctions.
[0031] The invention will be now described by means of non limiting examples referring to the following figures.
[0032] FIG. 1: a) SDS-PAGE of a representative set of purified recombinant human proteins stained with Coomassie blue; b) Western Blot analysis using an anti-histidine (His) tag monoclonal Antibody (mAb); molecular weight markers are shown on the left in kDa.
[0033] FIG. 2: a) Plot of the control human IgG curve. The dots correspond to the experimental average signal (Mean Fluorescence Intensity, y-axis) detected for each IgG concentration reported on the x-axis in nanograms per spot (ng/spot), while the continuous line corresponds to the interpolated resulting sigmoid curve. Dynamic Range: refers to a range of values that can be measured by a signal (i.e. Mean Fluorescence Intensity). Linear Range: is the range of concentration over which the signal intensity is linear, i.e. directly proportional to the spot concentration. b) Number of proteins detected with the anti-His mAb. About 90% of proteins were spotted with success on the slides (i.e. about 90% of the proteins produced signals that were significantly above the background signal). Histograms: Proteins were considered "Present" when at least two out of the four replicates gave a signal above the background, otherwise they were considered "Absent". c) The human protein microarray containing 1626 His-tagged recombinant proteins was probed with an anti-His mAb followed by a secondary α-mouse antibody Alexa-647-labeled. The whole microarray included 24 grids (as the one shown in the enlarged box) each one containing 304 proteins spots and including also positive (such as viral and bacterial proteins) and negative (such buffer or BSA) controls (positive Ctr and negative Ctr frames), as well as IgG calibration curve (HulgG curve) for data normalization. Proteins and controls were always spotted in quadruplicates, while IgG were spotted in eight replicates. d) Correlation among spot intensities of two different slides (Slide 1 Vs Slide 45) of the same batch. The scatter plot indicates a positive correlation. The correlation coefficient is 0.9, indicating a high reproducibility of the signals derived from the proteins spotted.
[0034] FIG. 3: Differences in immunoreactivity between serum samples. Representative image of the autoantigen microarrays incubated with a) sera from an AILD patient and b) a healthy donor (HD). c) Comparison of Mean Fluorescence Intensities (MFIs) of all spotted proteins against two sera populations: Healthy Donor (HD), Autoimmune Liver Diseases (AILD) patients. Each dot represents the MFI of a single protein. A cut-off value 4.000 was used to score a protein as positive. Asterisks: statistical significance, Student's t-test (pval <0.01). d) Percentage of antigens recognized by more than 15% of the sera tested; the threshold was determined on the basis of the average HD recognition (left bars), and the percentage of reactive sera for each population (i.e. sera reacting with more than 3% of the proteins screened; the threshold was determined on the basis of the average HD reactivity) (right bars). Asterisk; statistical significance (χ2 test (pval <0.01).
[0035] FIG. 4: a) Hierarchical Clustering of Training (upper panel) and Test set (lower panel) with regard to the 25 autoantigens selected as specifically recognized by AILD patients on the basis of i) higher recognition frequency and ii) higher mean fluorescence intensities as compared to healthy donors. Sera are represented in columns while proteins are in rows. Red indicates positive immunoreactivity, and blue low or no immunoreactivity; b) Bar graph of Mean Fluorescence intensities of the 25 autoantigens selected in Discovery sample set (Training and Test set). Significant differences were observed between AILD and HCV patients sera for seventeen autoantigens (into the box). Asterisks, p val <0.01 (Student's t-test). P.sup.(a): p-val of AILD versus Hepatitis C patients, P.sup.(b): p-val of AILD versus Healthy donors.
[0036] FIG. 5: Boxplots of DELFIA results for the 17 autoantigens tested. The signal distributions, after natural logarithm trasformation, are displayed. The boxes define the interquartile range (IQR). The extreme outliers beyond the 1.5 IQR+median are showed as dots. Three known AILD control proteins (AGPR-1, CYP2D6, PDH) (grey box) were also compared. HD: Helathy donors; AILD: autoimmune liver disease, VH: Viral hepatitis.
[0037] FIG. 6: Six proteins are confirmed as AIH autoantigens in DELFIA® assay. Recognition frequency of the best six autoantigens as determined by DELFIA®. Proteins were tested with sera from AILD patients (n. 50 AIH and n. 50 PBC), healthy donors (n. 50) and HBV or HCV viral hepatitis (VH) patients (n. 74). Each sera was tested 3 times in independent experiments. Asterisk: statistical significance (Fisher exact test, pval <0.01).
[0038] FIG. 7: Combination of the six autoantigens identifies AILD patients with high sensitivity and specificity. (a) Numbers in the boxes indicate AIH and HD sera that, in DELFIA® assay, recognize (positive) or do not recognize (negative) combination of six (i), four (ii) or three (iii) of the six autoantigens. Sensitivity (SE) and Specificity (SP) with 95% confidence intervals (C.I.) are indicated in the lower part of the panels. p-values are calculated with χ2 tests. (b) Logistic regression models for the six, four and three autoantigens (red curves) and the three known control proteins (AGPR1, CYP2D6, PDH) (black curves) were calculated and represented as ROC curves and corresponding values of Area Under the Curve (AUC).
Serum Samples
[0039] Samples used for this study were collected in five different hospitals: i) Policlinico Ospedale Maggiore, Transfusional Unit, Milan; ii) Hepatology Unit, University Hospital, Pisa, Italy; iii) Sant'Orsola-Malpighi University Hospital, Bologna, Italy; iv) Center for Autoimmune Liver Diseases, IRCCS Istituto Clinico Humanitas, Rozzano, Italy; v) Center for Systemic Manifestations of Hepatitis Viruses (MaSVE), University of Firenze, Italy. For the discovery phase, 218 sera were used (15 AIH, 15 PBC 78 HD, 110 HCV), while for the validation phase 224 sera were used (50 AIH, 50 PBC, 50 HD, 50 HCV, 24 HBV). Table 1 reports the clinical characteristics and ages of the patients and donors enrolled in this invention, for the microarray analysis (Example 2-3).
TABLE-US-00001 TABLE 1 Clinical characteristics of the serum samples used in this invention Age Sub Mean ± SD Sex Group group.sup.{circumflex over ( )} n. Source* (median) Gen (n.) Discovery Training Healthy Donors HD 39 1 44 ± 10 f(12) Set (46) m(27) Liver Autoimmune AIH 8 2 51 ± 20 f(8) disease (52) m(0) PBC 7 2 50 ± 12 f(6) (52) m(1) Test Set Healthy Donors HD 39 1, 2 44 ± 10 f(8) (44) m(31) Liver Autoimmune AIH 7 2 49 ± 23 f(5) disease (54) m(2) PBC 8 2 59 ± 22 f(8) (56) m(0) Viral hepatitis HCV 110 2, 3 55 ± 15 f(41) (56) m(69) Validation Healthy Donors HD 50 1 45 ± 9 f(8) (47) m(42) Liver Autoimmune disease AIH 50 2, 4 45 ± 21 f(41) (49) m(9) PBC 50 2, 4 nd ± nd (nd) f(nd) m(nd) Viral hepatitis HCV 50 5 52 ± 20 f(20) (52) m(30) HBV 24 2 51 ± 14 f(8) (52) m(16) *Origin of samples: (1) Transfusional Unit, Ospedale Maggiore Policlinico, Milan; (2) Sant'Orsola University Hospital, Bologna; (3) Hepatology Unit, University Hospital, Pisa; (4) Center for Autoimmune Liver Diseases, IRCCS Istituto Clinico Humanitas, Rozzano; (5) Center for Systemic Manifestations of Hepatitis Viruses (MaSVE), Firenze. {circumflex over ( )}HD: Healthy Donor; AIH: Autoimmune hepatitis; PBC: Primary Billiary Cirrhosis; HCV: Chronic C viral hepatitis patients; HBV:Chronic B viral hepatitis patients
Human Proteins: Selection, Expression and Purification
[0040] Genes whose translated products carry a secretion signal peptide or at least one transmembrane domain were selected, cloned and expressed in a high through-put system as histidine-tagged products as described (Grifantini R. et al., 2011, Journal of proteomics). 1626 full-length proteins or protein domains were expressed in E. coli and purified from the bacterial insoluble fraction by Immobilized metal ion affinity chromatography (IMAC, GE).
[0041] Human, viral or bacterial proteins were used as biological or technical controls in the microarray. In particular genes encoding for Core protein and Non-structural proteins NS3 (from HCV genotype 1), NS3-4a (from HCV genotype 2) NS5b (from HCV genotype 1), Tetanus toxin and H1N1 were subcloned in E. coli strain DH5α and expressed in BL21(DE3), respectively. Bovine Serum Albumin (BSA), Human Serum Albumin, Human Glutathione-S-Transferase and Protein A from Staphylococcus aureus were purchased from Sigma.
[0042] For DELFIA® experiments, Pyruvate DeHydrogenase (PDH) protein was purchased by Sigma, while genes encoding Cytochrome P450 2D6 (CYP2D6) and asialoglycoprotein receptor 1 (ASGR-1) from Ultimate® Human ORF Clones were purchased by Invitrogen and were subcloned in E. coli strain DH5α and expressed in BL21(DE3), respectively. All the corresponding proteins were purified by affinity chromatography on IMAC resin.
Protein Quality Control
[0043] Purified recombinant proteins (10-15 μg total protein), obtained as described above, were stored at 4° C. and analyzed by SDS-PAGE (Criterion PAGE system Bio-Rad) followed by Coomassie Blue staining of the gel immediately before spotting them, to assess their integrity, and purity level. Proteins showing purity levels >70% (ChemiDoc® XRS, Quantity One® software; Bio-Rad) were used for protein array preparation.
[0044] For Western blot analysis, aliquots (0.5 μg) of the proteins were resolved on 4-12% pre-cast SDS-PAGE gradient Tricine gels under reducing conditions, and electroblotted onto nitrocellulose membranes (Bio-Rad), according to the manufacturer's instructions. The membranes were blocked with 5% non-fat milk in 1×PBS plus 0.1% Tween 20 (TPBS) for 1 h at room temperature, incubated with the α-His mAb (GE-Healthcare) diluted 1:1000 in 3% non-fat milk in TPBS 0.1% for 1 h at room temperature, and washed three times in TPBS 0.1% (FIG. 1). The secondary HRP-conjugated antibody (α-mouse immunoglobulin/HRP, GE-Healthcare) was diluted 1:1000 in 3% non-fat milk in TPBS-0.1% and incubated for 1 h at room temperature. The proteins were visualized by enhanced chemiluminescence (Super Signal West Pico Chemiluminescence Substrate Thermo Scientific, USA) according to the manufacturer's specifications. Chemiluminescence was detected with an LAS-3000 (Fujifilm, USA).
Protein Microarray Printing
[0045] Protein MicroArrays were generated by spotting the 1626 affinity-purified recombinant proteins (0.5 mg/ml, in 6M Urea) in 4 replicates on nitrocellulose-coated slides (FAST slides, GE-Healthcare) using Stealth SMP3® spotting pins (TeleChem International, Sunnyvale, Calif.) and a Microgrid II microarray contact printer (Biorobotics), resulting in spots of approximately 130 μm in diameter. As experimental positive control, to assess the sensitivity and reproducibility of the arrays and for signal normalization, a curve of human IgG(s) at 11 different concentrations (solutions from 0.001 to 1 mg/ml) was spotted on the arrays in 8 replicates (in 6M Urea) and detected with Alexa-647 conjugated α-Human IgG secondary antibody (Invitrogen). As negative controls the spotting buffer alone, was printed and used to assess possible non-specific signals due to cross contamination.
[0046] A quality control of the spotting procedure was performed on 10% of randomly chosen slides, by confirming the presence of the total immobilized proteins using the α-His mAb, followed by detection using a Alexa-647 conjugated α-Human IgG secondary antibody (FIG. 2). The spotted microarrays were allowed to remain at room temperature for 1 h before storage at 4° C. until use.
Incubation and Scanning of Protein Microarray
[0047] Incubation was automatically performed with a TECAN Hybridization Station (HS4800® Pro; TECAN, Salzburg, Austria). The microarray slides were prewashed 3 min in TPBS 0.1% Tween 20, and saturated with BlockIt® Microarray Blocking Buffer (Arrayit Corporation) for 45 min at 25° C. under mild agitation. After injection of 105 μl of individual serum (diluted 1:300 in Blocking Buffer plus 0.1% Tween 20), incubation was performed at 25° C. for 45 min with low agitation. The microarrays were washed at 25° C. in TPBS for three cycles of 1 min wash time and 30 sec soak time.
[0048] Afterwards the microarray slides were incubated at 25° C. for 1 with Alexa-647 conjugated α-human IgG (Invitrogen) diluted at 1:800 in Blocking Buffer in the dark. The microarrays were again washed at 25° C. in TPBS for two cycles of 1 min wash time and 30 sec soak time, in PBS for two cycles of 1 min: 30 sec and finally in milliQ sterile water for one cycle of 15 sec.
[0049] The microarray slides were finally dried at 30° C. under nitrogen for 2 min, and scanned using a ScanArray Gx PLUS (PerkinElmer, Bridgeport Avenue Shelton, USA). 16-bit images were generated with ScanArray® software at 10 μm per pixel resolution and processed using ImaGene 8.0 software (Biodiscovery Inc, CA, USA). Laser of 635 nm was used to excite Alexa-647 dye. The fluorescence intensity of each spot was measured, signal-to-local-background ratios were calculated by ImaGene, and spot morphology and deviation from the expected spot position were considered using the default ImaGene settings.
Data Analysis
[0050] For each sample, the Mean Fluorescence Intensity (MFI) of replicated spots was determined, after subtraction of the background value for each spot, and subsequently normalized on the basis of the human IgG curve to allow comparison of data from different set of experiments (Bombaci M. et al., 2009, PLoS One). Briefly, the MFIs values of IgG, spotted at different concentrations, were best fitted by a sigmoid curve, using a maximum likelihood estimator (Harris J W et al., 1998, Handbook of Mathematics and Computational Science). The experimental average IgG curve of each slide was adjusted on the reference sigmoid IgG curve, and the background-subtracted MFI values of each protein were normalized accordingly. On the basis of these results, a normalized MFI value of 4.000 was chosen as the lowest signal threshold for scoring a protein as positively recognized by human sera. For each protein, a Coefficient of Variation (CV %), was calculated on the four replicate spots, for intra-assay reproducibility (Bombaci et al., 2009, PLoS One).
[0051] Recognition Frequency was defined as the percentage of sera reacting with a particular antigen in protein array with a MFI 4.000, and it was calculated for each group of sera. TIGR Multiexperiment Viewer (version MeV4.5) software (Saeed, A. I., et al., 2006, Methods in enzymology) was used to perform an unsupervised bi-dimensional hierarchical clustering.
Dissociation-Enhanced Lanthanide Fluorescence ImmunoAssay DELFIA® Assays
[0052] The DELFIA® assay is a time-resolved fluorescence method that can be used to study antibody binding to solid-phase proteins or peptides. The purified recombinant proteins were used at a concentration of 20 μg per milliliter (Frulloni L. et al., 2009, N Engl J Med) in 6 M urea to coat DELFIA® plates (PerkinElmer). Plates were then blocked for 1 hour at 37° C. with a blocking reagent (PerkinElmer). The blocking buffer was than discarded, and the serum samples were diluted in a 1:300 solution in phosphate buffered saline plus 1% bovine serum albumin (Sigma), plus 0.1% Tween 20 (Sigma) and incubated on the plates 1 hour at 37° C. Plates were then washed 5 times with washing buffer (PerkinElmer). Bound antibodies were detected with europium-labeled α-human IgG serum (1:500 in diluting buffer, PerkinElmer), incubated 30 min at RT in the dark. The wells were again washed in the same washing buffer. After a 10 min incubation at RT, the plates were read on a Infinite F200 PRO instrument (Tecan). Fluorescence intensity values higher than the mean of buffer plus 3 standard deviations were considered to be positive.
Statistical Analysis
[0053] Results of Protein Microarray and DELFIA® experiments from sera of patients and healthy donors were compared using the two-tailed χ2 test, the Student's t-test or the Fisher's exact tests. The ANOVA test was used for all the others analyses. The Benjamini-Hochberg correction for multiple testing was used for the analysis of microarray data. Statistical analysis was carried out with the use of GraphPad Prism 5 software, version 5.01. To evaluate the performance of autoantigens combinations in discriminating AILD patients from Healthy donors or HCV patients, logistic regression analysis was performed with R. We the signals of respectively 6, 4 or 3 selected autoantigens we created logistic regression models. The probabilities were calculated as follows: p=exp((Σ(bixi)+c)/(1+Σ(bixi)+c), where p is the probability of each case, i=1 to n; b is the regression coefficient of a given autoantigen, x is signal intensity and c is a constant generated by the model. ROCR package was used to obtain the ROC curves of the models and the Area Under Curve (AUC) values (Sing T. et al. 2005, Bioinformatics).
Example 1
Design and Development of a Human Protein Microarray
[0054] To study the serological profile of patients diagnosed with autoimmune liver diseases versus a panel of self proteins, the authors developed a protein array by printing 1626 human recombinant (see details in Materials and Methods section) products that corresponded to 1371 distinct human proteins distributed as shown in Table 2.
TABLE-US-00002 TABLE 2 Predicted subcellular localization of the human recombinant proteins for the microarray construction (Grifantini et al., 2011). Compartment % of proteins Cell membrane 46 Secreted 23 Intracell. Membrane 11 Cytoplasm 11 Mito. Membrane 4 Nucleus 4 Mithocondrion 1
[0055] Briefly, 1329 of the 1371 the proteins were first selected through a bioinformatic analysis of the whole human genome as translated sequences carrying i) signal peptides, ii) at least one transmembrane domain, iii) having unknown biological function. Fortytwo of the 1371 proteins had a well known immunological function, CD number assigned and were all surface exposed. The majority of printed human proteins were expressed as N-terminal His-tag fusions while 48 proteins where expressed as double tagged fusions, with an N-terminal glutathione S-transferase (GST) and with C-terminal Histidines. Proteins obtained after affinity purification from the bacterial insoluble fraction showed purity levels >70%, as estimated by densitometric scan of SDS-PAGE gels (see Materials and Methods) (FIG. 1a). Moreover, we performed western blot analysis on random protein samples with the anti-His mAb. About 80% of the probed proteins gave a positive signal. In addition for almost 90% of the latter proteins, the molecular weights of the bands detected by western blot corresponded to those observed by SDS-PAGE (FIG. 1b).
[0056] Protein arrays were prepared by printing onto nitrocellulose-covered glass slides four replicates of each protein. In addition, we included in the array, several biological and technical controls including human, viral and bacterial proteins (Materials and Methods). Replicates were randomly distributed to get optimal signal reproducibility. Moreover. eleven different amounts of human IgGs at a known concentration (from 8.24×10-4 to 7×10-1 ng of immobilized protein/spot) were printed also on the array in eight replicates (FIG. 2a).
[0057] The quality of the immobilized proteins on the arrays were determined by probing 10% of the slides with an anti-His mAb, and 89% of the proteins produced signals that were significantly above the background (FIG. 2b). The final protein array design consisted of 24 grids each of 304 spots, for a total of 7296 spots (FIG. 2c). The correlation among spot intensities of two different slides of the same batch indicates a high reproducibility of the signals derived from the proteins spotted (FIG. 2d).
Example 2
Patients Affected by Autoimmune Liver Diseases Show Higher Auto-Immunoreactivity as Compared to Healthy Donors
[0058] In an attempt to determine a panel of autoantigens differentially recognized by patients sera with AILD compared to healthy individuals (HD), the protein microarrays were probed with a sample set (defined as Training set as indicated in Table 1 in Materials and Methods) comprising 15 sera patients with AILD, and 39 sera from healthy donors. The clinical characteristics of each group of sera are summarized in Table 1.
[0059] Sera reactivity was evaluated by detecting total IgG bound to each protein spot using Alexa-647 conjugated α-human IgG and measuring the fluorescence intensity (FI) values for each protein. To compare data from different experiments, we used a normalization method, as previously described (Bombaci M et al., 2009, PLoS One). Briefly, the experimental average IgG curve of each slide was adjusted on a reference sigmoid IgG curve, and the background-subtracted mean fluorescence intensities (MFIs) values of each protein were normalized accordingly. On the basis of these results, a normalized-MFI value of 4000 was chosen as the lowest signal threshold for scoring a protein as positively recognized by human sera.
[0060] First of all, total reactivity of patients against healthy donors sera was evaluated. Representative images of a zoomed grid of the microarray probed with sera derived from healthy donors and AILD patients are shown in FIGS. 3a and b. As depicted in FIG. 3c, the strongest autoreactivity was observed against all the proteins present on the arrays in patients sera with AILD, but not in the control sera (HD group).
[0061] Significant divergences between patients and healthy donors were also observed in terms of the recognition frequencies, indicating differential protein-specific IgG levels FIG. 3d: in details, the percentage of proteins recognized by more than 15% of the tested sera with MFI>4000 was about 8% (132 proteins) in the case of both patients groups but only 3% (41 proteins) in the case of healthy donors (left). On the other hand, 53% of patients with AILD contain antibodies that react intensively with at least 3% of the proteins on the arrays, whereas only 18% of 39 controls had such antibodies (right panel). In both cases the differences observed between the reactivity of patients and healthy donors were statistically significant (chi-square test, p val <0.01).
Example 3
Selection and Identification of Specifically Recognized Autoantigens Profile
[0062] Having previously established that patients with AILD of Training set displayed an increased autoreactivity when compared to healthy donors, the authors selected the autoantigens specifically recognized by those patients. Following normalization, individual autoantigens from protein microarray were ranked according to (i) the recognition frequency and (ii) the mean fluorescence intensity of AILD patients as compared to the healthy donors.
[0063] To be considered of potential interest, the antigen-specific responses had to occur with a significantly higher signal intensity in patients sera than in healthy donors sera (T test's p val <0.01). In particular, the proteins should be recognized in less than 10% of the healthy donors sera and in at least 25% of sera from patients groups (Fisher test's pval <0.01). By using this approach, the authors identified 25 distinct proteins (Table 3) showing a higher immunoreactivity with AILD patients .compared to controls sera.
TABLE-US-00003 TABLE 3 Brief description of the human sequences listed SEQ ID Accession NO Prot_ID Protein Amino acid sequence NO: 1 YM0078 ENSP00000379111 PRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTYAVNIWSEN DPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTW SEWSPST NO: 2 YM0120 ENSP00000386923 SNWGCYGNIQSLDTPGASCGIGRRHGLNYCGVRASERLAEIDMP YLLKYQPMMQTIGQKYCMDPAVIAGVLSRKSPGDKILVNMGDRTS MVQDPGSQAPTSWISESQVSQTTEVLTTRIKEIQRRFPTWTPDQY LRGGLCAYSGGAGYVRSSQDLSCDFCNDVLARAKYLKRHGF NO: 3 YM0736 ENSP00000350556 MFVDNRIQKSMLLDLNKEIMNELGVTVVGDIIAILKHAKVVHRQDM CKAATESVPCSPSPLAGEIRRGTSAASRMITNSLNHDSPPSTPPR RPDTSTSKISVTVSNKMAAKSAKATAALARREEESLAVPAKRRRV TAEMEGKYVINMPKGTTPRTRKILEQQQAAKGLHRTSVFDRLGAE TKADTTTGSKPTGVFSRLGATPETDEDLAWDSDNDSSSSVLQYA GVLKKLGRGPAK NO: 4 YM1414 ENSP00000404259 SQGVCSKQTLVVPLHYNESYSQPVYKPYLTLCAGRRICSTYRTMY RVMWREVRREVQQTHAVCCQGWKKRHPGALTCEA NO: 5 YM1451 ENSP00000343084 HEAHKTSLSSWKHDQDWANVSNMTFSNGKLRVKGIYYRNADICS RHRVTSAGLTLQDLQLWCNLRSVARGQIPSTL NO: 6 YM1503 ENSP00000375259 GVAEFHMSLTVSCPDPTPSTDPQGRHNREPILGRDDDFMCKQVK FRMCVVGRDGNAQSSVRYTGPLYRRKIRTEFLFVVFLLETRELKP QVNKNVQGTRPS NO: 7 YM1535 ENSP00000288466 MVFVLTYMDPKGEVKKTHLHLASFSPSSEVSCFTNKAQAKNCSV EGCPSEWSSPRNLRSTKSIGTIRATGGCLCSGTVLHFPIPGSASQ ASL NO: 8 YM1602 XP_934154 ATGILICMTKNLESVHSIVLAHSCYHHENKPRPDCCFQQKIRDTKS KVELPRHAHARLTNPQLTHRSMKINDCCIKPLRFGVTCYAAFCDN N NO: 9 YM1651 ENSP00000343493 AREEEITPVVSIAYKVLEVFPKGRWVLITCCAPQPPPPITYSLCGTK NIKVAKKVVKTHEPASFNLNVTLKSSPDLLTYFCWASSTSGAHVD SARLQMHWELWSKPVSELRANFTLQDRGAGPRVEMICQASSGS PPITNSLIGKDGQVHLQQRPCHRQPANFSFLPSQTSDWFWCQAA NNANVQHSALTVVPPGGDQKMEDWQGPLESPILALPLYRSTRRL SEEEFGGFRIGNGE NO: 10 YM1652 ENSP00000413076 VAKKVVKTHEPASFNLNVTLKSSPDLLTYFCWASSTSGAHVDSAR LQMHWELWSKPVSELRANFTLQDRGAGPRVEMICQASSGSPPIT NSLIGKDGQVHLQQRPCHRQPANFSFLPSQTSDWF NO: 11 YM1672 ENSP00000315731 AQYSSDRCSWKGSGLTHEAHRKEVEQVYLRCAAGAVEWMYPTG ALIVNLRPNTFSPARHLTVCIRSFTDSSGANIYLEKTGELRLLVPDG DGRPGRVQCFGLEQGGLFVEATPQQDIGRRTTGFQYELVRRHR ASDLHELSAPCRPCSDTEVLLAVCTSDFAVRGSIQQVTHEPERQD SAIHLRVSRLYRQKSRVFEPVPEGDGHWQGRVRTLLECGVRPGH GDFLFTGHMHFGEAR NO: 12 YM1708 ENSP00000392824 MFGVLEGAQANSENWIAPSGPWALGLWSSLYFLLFSTLEGRGGR VLSQSCSMAVAAASWISRENARSVKRSYMQSSPQRPKEPRNQR TSHTTPVC NO: 13 YM1801 ENSP00000367965 SGFTALHWAAKSGDGEMALQLVEVARRSGAPVDVNARSHGGYT PLHLAALHGHEDAAVLLVVRLGAQVHVRDHSGRRAYQYLRPGSS YALRRLLGDPGLRGTTEPDATGGGSGSLAARRPVQVAATILSSTT SAFLGVLADDLMLQDLARGLKKSSSFSKFLSASPMAPRKKTKIRG GLPAFSEISRRPTPGPLAGLVPSFPPTT NO: 14 YM1882 ENSP00000395006 SLIVFMEQVHRGIKGLVRDSHGKGIPNAIISVEGINHDIRTANDGDY WRLLNPGEYVVTAKAEGFTASTKNCMVGYDMGATRCDFTLSKTN MARIREIMEKFGKQPVSLPARRLKLRGQKRRQRG NO: 15 YM1980 ENSP00000362968 GSLSPTKYNLLELKESCIRNQDCETGCCQRAPDNCESHCAEKGS EGSLCQTQVFFGQYRACPCLRNLTCIYSKNEKWLSIAYGRCQKIG RQKLAKKMFF NO: 16 YM1989 ENSP00000415977 SQLELIDLSSNPFHCDCQLLPLHRWLTGLNLRVGATCATPPNARG QRVKAAAAVFEDCPGWAARKAKRTPASRPSARRTPIKGRQCGA DKVGHGAGGV NO: 17 YM2046 ENSP00000360191 GEAGGSCLRWEPHCQQPLPDRVPSTAILPPRLNGPWISTGCEVR PGPEFLTRAYTFYPSRLFRAHQFYYEDPFCGEPAHSLLVKGKVRL RRASWVTRGATEADYHLHKVGIV NO: 18 YM2213 ENSP00000414589 ATKLVTCPAPRQFAVGAFTAAGRAWLFAPSLGASFSKLRSQQRS RDFRGRLFLRAERRAGGFTS NO: 19 YM2273 ENSP00000374868 SSNLEGRTKSVIRQTGSSAEITCDLAEGSTGYIHWYLHQEGKAPQ RLLYYDSYTSSVVLESGISPGKYDTYGSTRKNLRMILRNLIENDSG VYYCATWDGHSDSDPPYTTLKTCLVAASPREEGMRWALLVLLAF LSPIPAPPTVFCARDHFLVEWDLSFERIFYSFSSGPFPSKAPERKA CSQKSSNLEGRMKSVTRPTGSSAEITCDLTVINAVYIHWYLQQEG KTPQHLLHYDV NO: 20 YM2279 ENSP00000374896 AGVTQTPKFHVLKTGQSMTLLCAQDMNHEYMYRYRQDPGKGLR LIYYSVAAALTDKGEVPNGYNVSRSNTEDFPLKLESAAPSQTSVY FCASSYSTALQGCLLSAHKGKGRCCPPPPPKTQGCPVQRSLHQE PWNPEWPQVARTV NO: 21 YM2315 ENSP00000374919 AKVTQTPGHLVKGKGQKTKMDCTPEKGHTFVYWYQQNQNKEFM LLISFQNEQVLQETEMHKKRFSSQCPKNAPCSLAILSSEPGDTALY LCASSQSTALKCQFLLAHKLVTDPAQEAGDVLGWKGVTENNWSQ LKPQCNLTQG NO: 22 YM2671 ENSP00000327628 RDKMRMQRIKVCEKRPSIDLCIHHCSYFQKCETNKICCSAFCGNIC MSIL NO: 23 YM2741 ENSP00000363414 PERWFPGSCHVFGQGHQLFHIFLVLCTLAQLEAVALDYEARRPIY EPLHTHWPHN NO: 24 YM2779 ENSP00000395093 QLLMYQQHTSHYDLERKGGYLMLSFIDFCPFSVMRLRSLPSPQR YTRQERYRARPPRVLERSGFHNENSLAIYQGLVYYLLWLHSVYDK PYADPVHDPTWRWWANNKQDQDYYFFLASNWRSAGGVSIEMD SYEKIYNLESAYELPERIFLDKGTEYSFAIFLSAQGHSFRTQSELGT AFQLHSQVDVGVVLADPGCIEASVKQEVLINRNSVLFSITLKDKKL CYDQGISGHHLME NO: 25 YM2814 ENSP00000258969 VVEELKLSHNPLKSIPDNAFQSFGRYLETLWLDNTNLEKFSDGAFL GVTTLKHVHLENNRLNQLPSNFPFDSLETLALTNNPWKCTCQLRG LRRWLEAKASRPDATCASPAKFKGQHIRDTDAFRSCKFPTKRSK KAGRH
[0064] In order to confirm the above results, a different set of proteins spotted in the microarray was used. This microarray now included all proteins identified as more reactive in the training set including the 24 proteins as indicated above (Table 4).
TABLE-US-00004 TABLE 4 Proteins printed on the focused microarray. Protein # Accession Protein Description Array_Id 1 ENSP00000292144 CD3g molecule, gamma (CD3-TCR complex) YM0066 2 ENSP00000418364 CD80 molecule YM0071 3 ENSP00000377248 CD86 molecule YM0072 4 ENSP00000228434 CD69 molecule YM0073 5 ENSP00000216223 interleukin 2 receptor, beta YM0076 6 ENSP00000379111 interleukin 4 receptor YM0078 7 ENSP00000345501 integrin, beta 7 YM0079 8 ENSP00000390133 transferrin receptor (p90, CD71) YM0083 9 ENSP00000262817 Transmembrane protein 59-like Precursor YM0099 10 ENSP00000361001 EF-hand domain-containing protein KIAA0494 YM0100 11 ENSP00000363041 CDGSH iron sulfur domain-containing protein 1 YM0107 12 ENSP00000296955 Discoidin, CUB and LCCL domain-containing protein YM0117 1 Precursor 13 ENSP00000386923 Lysozyme g-like protein 1 Precursor YM0120 14 ENSP00000302046 Ribonuclease K6 Precursor YM0125 15 ENSP00000317385 GLIPR1-like protein 2 YM0724 16 ENSP00000334044 Ubiquitin-like protein 4B YM0728 17 ENSP00000350556 Uncharacterized protein C19orf47 YM0736 18 ENSP00000312599 Transmembrane protein 70, mitochondrial Precursor YM0757 19 ENSP00000334308 Nesprin-3 YM0838 20 ENSP00000258436 Major facilitator superfamily domain-containing YM0872 protein 9 21 ENSP00000357480 Nogo-B receptor Precursor YM0887 22 ENSP00000412308 Uncharacterized protein C18orf19 YM0908 23 ENSP00000364502 Protein FAM70B YM0972 24 ENSP00000294258 Zinc finger protein-like 1 YM1020 25 ENSP00000262262 CD33 molecule YM1046 26 ENSP00000364621 Sushi domain-containing protein 3 YM1073 27 ENSP00000266943 Solute carrier family 46 member 3 Precursor YM1077 28 ENSP00000292301 chemokine (C-C motif) receptor 2 YM1099 29 ENSP00000317300 Lysophosphatidylcholine acyltransferase 4 YM1129 30 ENSP00000326267 EF-hand domain-containing protein C14orf143 YM1131 31 ENSP00000356048 Synaptotagmin-like protein 3 YM1134 32 ENSP00000342493 Regulator of microtubule dynamics protein 3 YM1183 33 ENSP00000352601 Low-density lipoprotein receptor-related protein YM1204 10 Precursor 34 ENSP00000347152 Attractin-like protein 1 Precursor YM1208 35 ENSP00000416040 UPF0606 protein KIAA1549 YM1214 36 ENSP00000248668 Leucine-rich repeat and fibronectin type III YM1217 domain- containing protein 1 Precursor 37 ENSP00000357631 Transcription elongation regulator 1-like protein YM1340 38 ENSP00000311657 GRAM domain-containing protein 2 YM1342 39 ENSP00000360822 Potassium channel subfamily T member 1 YM1343 40 ENSP00000418985 Solute carrier family 22 member 23 YM1351 41 ENSP00000311307 Uncharacterized protein C4orf26 Precursor YM1401 42 ENSP00000404259 EGF-like domain-containing protein 8 Precursor YM1414 43 ENSP00000384553 Zinc/RING finger protein 3 Precursor YM1423 44 ENSP00000343084 Putative uncharacterized protein YM1451 45 ENSP00000374125 Sialic acid-binding Ig-like lectin 15 Precursor YM1477 46 ENSP00000375259 Putative uncharacterized protein DKFZp667F0711 YM1503 Fragment 47 ENSP00000263569 Platelet receptor Gi24 Precursor YM1526 48 ENSP00000247618 Kinesin light chain 1 YM1529 49 ENSP00000368502 Secretoglobin-like protein Precursor YM1534 50 ENSP00000288466 zinc finger protein 618 YM1535 51 ENSP00000359571 Uncharacterized protein CXorf66 Precursor YM1536 52 ENSP00000315554 Spermatid maturation protein 1 YM1537 53 ENSP00000415978 Protein FAM74A1/A2 YM1542 54 ENSP00000325525 similar to Killer cell immunoglobulin-like receptor YM1546 3DL2 precursor (MHC class I NK cell receptor) (Natural killer associated transcript 4) (NKAT-4) (p70 natural killer cell receptor clone CL-5) 55 ENSP00000406884 HCG2023280cDNA FLJ30064 fis, clone YM1587 ADRGL2000323; 56 NP_001034884 Putative uncharacterized protein YM1593 57 ENSP00000374867 hypothetical protein LOC648852 YM1594 58 ENSP00000397690 Putative uncharacterized protein UNQ6190/ YM1599 PRO20217 Precursor 59 XP_934154 Putative uncharacterized protein YM1602 60 ENSP00000343493 Uncharacterized protein C17orf99 Precursor YM1651 61 ENSP00000413076 Uncharacterized protein C17orf99 Precursor YM1652 62 ENSP00000368396 UPF0631 protein HSD24 YM1668 63 ENSP00000328061 Uncharacterized protein C17orf74 YM1671 64 ENSP00000315731 Meteorin-like protein Precursor YM1672 65 ENSP00000331466 Transmembrane protein 95 Precursor YM1674 66 ENSP00000331466 Transmembrane protein 95 Precursor YM1675 67 ENSP00000304670 RING finger and transmembrane domain-containing YM1684 protein 1 68 ENSP00000303437 t-SNARE domain-containing protein 1 YM1693 69 ENSP00000392824 LP5624 YM1708 70 ENSP00000346747 Putative uncharacterized protein YM1720 71 ENSP00000262424 Cysteine-rich secretory protein LCCL domain- YM1770 containing 2 Precursor 72 ENSP00000360577 Enoyl-CoA hydratase domain-containing protein YM1778 2, mitochondrial Precursor 73 ENSP00000367965 Ankyrin repeat domain-containing protein 43 YM1801 Precursor 74 ENSP00000354240 RPE-spondin Precursor YM1810 75 OTTHUMP00000028326 Putative uncharacterized protein YM1824 76 ENSP00000395006 Carboxypeptidase-like protein X2 Precursor YM1882 77 ENSP00000298966 UPF0443 protein C11orf75 YM1909 78 ENSP00000350961 Transmembrane protein C9orf123 YM1914 79 ENSP00000378605 DnaJ homolog subfamily C member 30 YM1925 80 OTTHUMP00000028097 Putative uncharacterized protein YM1957 81 ENSP00000368662 Uroplakin-3-like protein Precursor YM1960 82 ENSP00000405827 Putative uncharacterized protein YM1971 83 ENSP00000362968 Colipase-like protein C6orf127 Precursor YM1980 84 ENSP00000409535 C-type lectin domain family 18 member B YM1985 Precursor 85 ENSP00000415977 Chondroadherin-like protein Precursor YM1989 86 ENSP00000412448 YM2013 87 ENSP00000414449 YM2036 88 ENSP00000360191 Protein APCDD1-like Precursor YM2046 89 ENSP00000321517 Putative uncharacterized protein YM2054 90 ENSP00000339578 Seven transmembrane helix receptor YM2071 91 ENSP00000397696 HSAL5836 YM2094 92 ENSP00000366415 Putative uncharacterized protein YM2100 93 ENSP00000345107 Putative uncharacterized protein YM2110 94 ENSP00000344029 Putative uncharacterized protein UNQ9165/ YM2111 PRO28630 Precursor 95 ENSP00000409458 Putative uncharacterized protein YM2136 96 ENSP00000398103 Putative uncharacterized protein UNQ6493/ YM2140 PRO21345 97 ENSP00000372305 Putative uncharacterized protein YM2144 98 ENSP00000411889 Putative uncharacterized protein UNQ6490/ YM2151 PRO21339 Precursor 99 ENSP00000349132 Protein FAM75A3 YM2168 100 ENSP00000406884 HCG2023280cDNA FLJ30064 fis, clone YM2202 ADRGL2000323; 101 ENSP00000414589 VGSA5840 YM2213 102 ENSP00000389279 AHPA9419 YM2214 103 ENSP00000358798 Calcium homeostasis modulator protein 3 YM2223 104 ENSP00000374897 YM2237 105 ENSP00000373765 monooxygenase, DBH-like 2 pseudogene (MOXD2) YM2250 106 ENSP00000374869 YM2251 107 ENSP00000304930 Sclerostin domain-containing protein 1 YM2255 Precursor 108 ENSP00000409076 Uncharacterized protein KIAA1644 Precursor YM2271 109 ENSP00000374868 Putative uncharacterized protein YM2273 ENSP00000374868 110 ENSP00000374880 YM2276 111 ENSP00000374896 Putative uncharacterized protein YM2279 ENSP00000374896 112 ENSP00000400516 Hematopoietic cell signal transducer Precursor YM2284 113 ENSP00000382000 CMT1A duplicated region transcript 15 protein- YM2286 like protein 114 ENSP00000311857 YM2289 115 ENSP00000331418 YM2297 116 ENSP00000374919 Putative uncharacterized protein YM2315 ENSP00000374919 117 OTTHUMP00000076641 Putative uncharacterized protein YM2322 118 OTTHUMP00000076959 Putative uncharacterized protein YM2326 119 ENSP00000222033 Zinc/RING finger protein 4 Precursor YM2335 120 ENSP00000251473 Lipid phosphate phosphatase-related protein YM2355 type 2 121 ENSP00000291934 Transmembrane protein 190 Precursor YM2388 122 ENSP00000393015 Serine protease 23 Precursor YM2441 123 ENSP00000414523 Leucine-rich repeat transmembrane neuronal YM2448 protein 1 Precursor 124 ENSP00000363345 Thymic stromal cotransporter homolog YM2453 125 ENSP00000307164 Prostate and testis expressed protein 1 YM2511 Precursor 126 ENSP00000355243 Protocadherin-7 Precursor YM2570 127 ENSP00000264661 Potassium voltage-gated channel subfamily H YM2576 member 4 128 ENSP00000266646 Inhibin beta E chain Precursor YM2613 129 ENSP00000344847 A disintegrin and metalloproteinase with YM2616 thrombospondin motifs 12 Precursor 130 ENSP00000298690 Ribonuclease 7 Precursor YM2626 131 ENSP00000321345 interleukin 23 receptor YM2646 132 ENSP00000361735 WAP four-disulfide core domain protein 8 YM2665 Precursor 133 ENSP00000327628 Protein WFDC10B Precursor YM2671 134 ENSP00000259216 Cryptic protein Precursor YM2707 135 ENSP00000349131 R-spondin-3 Precursor YM2726 136 ENSP00000347451 Leucine-rich repeat and immunoglobulin-like YM2728 domain- containing nogo receptor-interacting protein 1 Precursor 137 ENSP00000363414 Membrane progestin receptor alpha YM2741 138 ENSP00000385766 Leucine-rich repeat-containing protein 32 YM2767 Precursor 139 ENSP00000395093 Uncharacterized protein C19orf15 Precursor YM2779 140 ENSP00000258969 Chondroadherin Precursor YM2814 141 ENSP00000272134 left-right determination factor 1 YM2826 142 ENSP00000215885 Group 3 secretory phospholipase A2 Precursor YM2831 143 ENSP00000255039 Hyaluronan and proteoglycan link protein 2 YM2843 Precursor 144 ENSP00000380417 Interleukin-25 Precursor YM2854
[0065] In order to confirm the above results, the authors probed the focused protein microarray with a second serum sample set (Test Set as indicated in Table 1 Material and Methods) comprising other 15 sera of patients with AILD and 39 serum samples from healthy subjects. Following the same normalization and using criteria described above (see Materials and Methods), the authors confirmed that the 25 autoantigens were differentially recognized by patients compared to healthy donors with statistical significance. Interestingly, in unsupervised hierarchical clustering analysis these autoantigens allowed for good discrimination of the two populations of sera in both sample sets, as shown in FIG. 4a.
[0066] Having identified 25 autoantigens highly recognized by AILD patients, the authors asked whether non-autoimmune liver disease patients had an overlapped recognition pattern. They therefore tested the same microarray with sera from 110 patients with chronic HCV infection (Table 1). FIG. 4b shows the MFIs of the 25 autoantigens in each group of individuals (AILD, HD and HCV respectively), and highlights that, although some reactivity is observed in patients affected from HCV, 17 auto-antigens react preferentially and more strongly with sera from AILD patients, with statistical significance (p val <0.01).
Example 4
Validation of Selected Autoantigens with an Independent Sample Set Confirms that 6 Proteins are New Potential AILD Biomarkers
[0067] After having identified a total of 17 auto-antigens specific for autoimmune patients by protein microarray, the authors validated the results obtained by using the Dissociation-Enhanced Lanthanide Fluorescence ImmunoAssay method (DELFIA®) assay method (Materials and Methods).
[0068] By this assay the authors screened an independent sample set (Validation set as indicated in Table 1, Materials and Methods) comprising 100 AILD patients (50 AIH and 50 PBC), 50 healthy donors and 74 patients with chronic viral hepatitis (50 HCV and 24 HBV) measured by time-resolved fluorescence. All sera were tested at a dilution of 1:300 as described in Materials and Methods, and the antigen-specific IgG responses to each of the 17 selected autoantigens was measured by time-resolved fluorescence. Reproducibility of the results was confirmed using duplicate sample of selected sera.
[0069] All 17 antigens displayed higher mean fluorescence intensity compared to HD and chronic viral hepatitis (FIG. 5). Fluorescence intensity values higher than the mean plus three standard deviations of buffer signals among healthy donors were considered positive. As shown in Table 5, 6 of the 17 antigens displayed significantly higher recognition frequency by AILD patients compared to healthy donors, and were also significant when compared to viral hepatitis patients (FIG. 6), thus being considered as top candidates.
TABLE-US-00005 TABLE 5 Recognition frequencies of individual antigens identified as candidate AILD biomarkers in the validation sample set. Positive sera % HD AILD VH Description Prot. Id ComboA (n = 50) (n = 100) (n = 74) Asialoglyco- AGPR 50 70 53 protein receptor Cytochrome Cyp450 56 72 55 P4502D6 Pyruvate PDH 0 55 4 dehydrogenase Interleukin-4 YM0078 x∘• 2 74 14 receptor domain Putative YM1503 x 6 48 14 uncharacterized protein Putative YM1602 x 8 44 14 uncharacterized protein Uncharacterized YM1652 x∘ 4 47 15 protein C17orf99 Meteorin-like YM1672 x∘• 0 48 22 protein Precursor Protein APCDD1- YM2046 x∘• 2 63 28 like Precursor HD: Healthy donors; AILD: autoimmune liver disease patients, VH: Viral hepatitis patients. AAntigens used for combination assays comprising 6 (x), 4 (∘) and 3 (•) markers respectively. SE % = percentage of positive AIH sera; SP % = percentage of negative HD or VH sera.
[0070] These top 6 antigens, showed high sensitivity (from 44 to 74% of positive AILD patients) and specificity (from 92 to 100% of negative HD). Interestingly, individual sensitivity was comparable to that obtained in our hands by 3 known AILD markers, CYP2D6 (Cytochrome Peroxidase 2D6), AGPR-1 (Asialo-Glycoprotein Receptor 1) and PDH (Pyruvate DeHydrogenase), (Jensen, D M, 1978, The New England journal of medicine; Van de Water J. et al., 1993, The Journal of clinical investigation), while individual specificity was far better for our candidates compared to the benchmarks.
[0071] The authors next assessed the discrimination power of combinations of the six autoantigens. We therefore tested combinations of three (YM0078, YM1672, YM2046), four (YM0078, YM1652, YM1672, YM2046) or six proteins (YM1672, YM0078, YM2046, YM1652, YM1503, YM1602). FIG. 7a shows that combinations allow clear discrimination between patients with AILD and healthy donors, with sensitivities of 77%±4 (86, 81 and 79%, for 3, 4 and 6 combos) and specificities of 91%±8 (96, 92 and 82% respectively). Moreover, FIG. 7b shows the ROC curves of logistic regression models obtained with the same combinations of new antigens compared to the combination of the three known control proteins, and illustrates that any of the new antigens combo is superior to the combo of the three known control proteins.
[0072] 1. Czaja, A. J., and Manns, M. P. 2010. Gastroenterology 139:58-72 e54.
[0073] 2. Makol, A., Watt, K. D., and Chowdhary, V. R. 2011. 2011:390916.
[0074] 3. Bogdanos D P, Mieli-Vergani G, Vergani D. 2009. 29(3):241-53.
[0075] 4. Selmi, C., Mackay, I. R., and Gershwin, M. E. 2011. 89:70-80.
[0076] 5. Muratori L, Muratori P, Granito A, Pappas G, Cassani F, Lenzi M. 2010. Dig Liver Dis 42(11):757-64.
[0077] 6. Muratori, L., Granito, A., Muratori, P., Pappas, G., and Bianchi, F. B. 2008. Clinics in liver disease 12:261-276; vii.
[0078] 7. Bogdanos, D. P., Invernizzi, P., Mackay, I. R., and Vergani, D. 2008. World journal of gastroenterology: WJG 14:3374-3387.
[0079] 8. Vergani, D., Alvarez, F., Bianchi, F. B., Cancado, E. L., Mackay, I. R., Manns, M. P., Nishioka, M., and Penner, E. 2004. Journal of hepatology 41:677-683.
[0080] 9. Zachou, K., Rigopoulou, E., and Dalekos, G. N. 2004. Journal of autoimmune diseases 1:2.
[0081] 10. Song, Q., Liu, G., Hu, S., Zhang, Y., Tao, Y., Han, Y., Zeng, H., Huang, W., Li, F., Chen, P., et al. 2010. 9:30-39.
[0082] 11. Grifantini, R., Pagani, M., Pierleoni, A., Grandi, A., Parri, M., Campagnoli, S., Pileri, P., Cattaneo, D., Canidio, E., Pontillo, A., et al. 2011. Journal of proteomics 75:532-547.
[0083] 12. Bombaci, M., Grifantini, R., Mora, M., Reguzzi, V., Petracca, R., Meoni, E., Balloni, S., Zingaretti, C., Falugi, F., Manetti, A. G., et al. 2009. PloS one 4:e6332.
[0084] 13. Harris, J. W. and Stocker, H.1998. Handbook of Mathematics and Computational Science. New York: Springer-Verlag.
[0085] 14. Saeed, A. I., Bhagabati, N. K., Braisted, J. C., Liang, W., Sharov, V., Howe, E. A., Li, J., Thiagarajan, M., White, J. A., and Quackenbush, J. 2006. Methods in enzymology 411:134-193.
[0086] 15. Frulloni, L., Lunardi, C., Simone, R., Dolcino, M., Scattolini, C., Falconi, M., Benini, L., Vantini, I., Corrocher, R., and Puccetti, A. 2009. The New England journal of medicine 361:2135-2142.
[0087] 16. Sing, T., Sander, O., Beerenwinkel, N., and Lengauer, T. 2005. ROCR: visualizing classifier performance in R. Bioinformatics 21:3940-3941.
[0088] 17. Jensen, D. M., McFarlane, I. G., Portmann, B. S., Eddleston, A. L., and Williams, R. 1978. The New England journal of medicine 299:1-7.
[0089] 18. Van de Water, J., Turchany, J., Leung, P. S., Lake, J., Munoz, S., Surh, C. D., Coppel, R., Ansari, A., Nakanuma, Y., and Gershwin, M. E. 1993. The Journal of clinical investigation 91:2653-2664.
Sequence CWU
25197PRTHomo sapiens 1Pro Arg Ala Pro Gly Asn Leu Thr Val His Thr Asn Val Ser Asp Thr 1 5 10 15 Leu Leu Leu Thr Trp Ser Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr 20 25 30 Asn His Leu Thr Tyr Ala Val Asn Ile Trp Ser Glu Asn Asp Pro Ala 35 40 45 Asp Phe Arg Ile Tyr Asn Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile 50 55 60 Ala Ala Ser Thr Leu Lys Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg 65 70 75 80 Ala Trp Ala Gln Cys Tyr Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser 85 90 95 Thr 2175PRTHomo sapiens 2Ser Asn Trp Gly Cys Tyr Gly Asn Ile Gln Ser Leu Asp Thr Pro Gly 1 5 10 15 Ala Ser Cys Gly Ile Gly Arg Arg His Gly Leu Asn Tyr Cys Gly Val 20 25 30 Arg Ala Ser Glu Arg Leu Ala Glu Ile Asp Met Pro Tyr Leu Leu Lys 35 40 45 Tyr Gln Pro Met Met Gln Thr Ile Gly Gln Lys Tyr Cys Met Asp Pro 50 55 60 Ala Val Ile Ala Gly Val Leu Ser Arg Lys Ser Pro Gly Asp Lys Ile 65 70 75 80 Leu Val Asn Met Gly Asp Arg Thr Ser Met Val Gln Asp Pro Gly Ser 85 90 95 Gln Ala Pro Thr Ser Trp Ile Ser Glu Ser Gln Val Ser Gln Thr Thr 100 105 110 Glu Val Leu Thr Thr Arg Ile Lys Glu Ile Gln Arg Arg Phe Pro Thr 115 120 125 Trp Thr Pro Asp Gln Tyr Leu Arg Gly Gly Leu Cys Ala Tyr Ser Gly 130 135 140 Gly Ala Gly Tyr Val Arg Ser Ser Gln Asp Leu Ser Cys Asp Phe Cys 145 150 155 160 Asn Asp Val Leu Ala Arg Ala Lys Tyr Leu Lys Arg His Gly Phe 165 170 175 3237PRTHomo sapiens 3Met Phe Val Asp Asn Arg Ile Gln Lys Ser Met Leu Leu Asp Leu Asn 1 5 10 15 Lys Glu Ile Met Asn Glu Leu Gly Val Thr Val Val Gly Asp Ile Ile 20 25 30 Ala Ile Leu Lys His Ala Lys Val Val His Arg Gln Asp Met Cys Lys 35 40 45 Ala Ala Thr Glu Ser Val Pro Cys Ser Pro Ser Pro Leu Ala Gly Glu 50 55 60 Ile Arg Arg Gly Thr Ser Ala Ala Ser Arg Met Ile Thr Asn Ser Leu 65 70 75 80 Asn His Asp Ser Pro Pro Ser Thr Pro Pro Arg Arg Pro Asp Thr Ser 85 90 95 Thr Ser Lys Ile Ser Val Thr Val Ser Asn Lys Met Ala Ala Lys Ser 100 105 110 Ala Lys Ala Thr Ala Ala Leu Ala Arg Arg Glu Glu Glu Ser Leu Ala 115 120 125 Val Pro Ala Lys Arg Arg Arg Val Thr Ala Glu Met Glu Gly Lys Tyr 130 135 140 Val Ile Asn Met Pro Lys Gly Thr Thr Pro Arg Thr Arg Lys Ile Leu 145 150 155 160 Glu Gln Gln Gln Ala Ala Lys Gly Leu His Arg Thr Ser Val Phe Asp 165 170 175 Arg Leu Gly Ala Glu Thr Lys Ala Asp Thr Thr Thr Gly Ser Lys Pro 180 185 190 Thr Gly Val Phe Ser Arg Leu Gly Ala Thr Pro Glu Thr Asp Glu Asp 195 200 205 Leu Ala Trp Asp Ser Asp Asn Asp Ser Ser Ser Ser Val Leu Gln Tyr 210 215 220 Ala Gly Val Leu Lys Lys Leu Gly Arg Gly Pro Ala Lys 225 230 235 479PRTHomo sapiens 4Ser Gln Gly Val Cys Ser Lys Gln Thr Leu Val Val Pro Leu His Tyr 1 5 10 15 Asn Glu Ser Tyr Ser Gln Pro Val Tyr Lys Pro Tyr Leu Thr Leu Cys 20 25 30 Ala Gly Arg Arg Ile Cys Ser Thr Tyr Arg Thr Met Tyr Arg Val Met 35 40 45 Trp Arg Glu Val Arg Arg Glu Val Gln Gln Thr His Ala Val Cys Cys 50 55 60 Gln Gly Trp Lys Lys Arg His Pro Gly Ala Leu Thr Cys Glu Ala 65 70 75 576PRTHomo sapiens 5His Glu Ala His Lys Thr Ser Leu Ser Ser Trp Lys His Asp Gln Asp 1 5 10 15 Trp Ala Asn Val Ser Asn Met Thr Phe Ser Asn Gly Lys Leu Arg Val 20 25 30 Lys Gly Ile Tyr Tyr Arg Asn Ala Asp Ile Cys Ser Arg His Arg Val 35 40 45 Thr Ser Ala Gly Leu Thr Leu Gln Asp Leu Gln Leu Trp Cys Asn Leu 50 55 60 Arg Ser Val Ala Arg Gly Gln Ile Pro Ser Thr Leu 65 70 75 6101PRTHomo sapiens 6Gly Val Ala Glu Phe His Met Ser Leu Thr Val Ser Cys Pro Asp Pro 1 5 10 15 Thr Pro Ser Thr Asp Pro Gln Gly Arg His Asn Arg Glu Pro Ile Leu 20 25 30 Gly Arg Asp Asp Asp Phe Met Cys Lys Gln Val Lys Phe Arg Met Cys 35 40 45 Val Val Gly Arg Asp Gly Asn Ala Gln Ser Ser Val Arg Tyr Thr Gly 50 55 60 Pro Leu Tyr Arg Arg Lys Ile Arg Thr Glu Phe Leu Phe Val Val Phe 65 70 75 80 Leu Leu Glu Thr Arg Glu Leu Lys Pro Gln Val Asn Lys Asn Val Gln 85 90 95 Gly Thr Arg Pro Ser 100 792PRTHomo sapiens 7Met Val Phe Val Leu Thr Tyr Met Asp Pro Lys Gly Glu Val Lys Lys 1 5 10 15 Thr His Leu His Leu Ala Ser Phe Ser Pro Ser Ser Glu Val Ser Cys 20 25 30 Phe Thr Asn Lys Ala Gln Ala Lys Asn Cys Ser Val Glu Gly Cys Pro 35 40 45 Ser Glu Trp Ser Ser Pro Arg Asn Leu Arg Ser Thr Lys Ser Ile Gly 50 55 60 Thr Ile Arg Ala Thr Gly Gly Cys Leu Cys Ser Gly Thr Val Leu His 65 70 75 80 Phe Pro Ile Pro Gly Ser Ala Ser Gln Ala Ser Leu 85 90 892PRTHomo sapiens 8Ala Thr Gly Ile Leu Ile Cys Met Thr Lys Asn Leu Glu Ser Val His 1 5 10 15 Ser Ile Val Leu Ala His Ser Cys Tyr His His Glu Asn Lys Pro Arg 20 25 30 Pro Asp Cys Cys Phe Gln Gln Lys Ile Arg Asp Thr Lys Ser Lys Val 35 40 45 Glu Leu Pro Arg His Ala His Ala Arg Leu Thr Asn Pro Gln Leu Thr 50 55 60 His Arg Ser Met Lys Ile Asn Asp Cys Cys Ile Lys Pro Leu Arg Phe 65 70 75 80 Gly Val Thr Cys Tyr Ala Ala Phe Cys Asp Asn Asn 85 90 9237PRTHomo sapiens 9Ala Arg Glu Glu Glu Ile Thr Pro Val Val Ser Ile Ala Tyr Lys Val 1 5 10 15 Leu Glu Val Phe Pro Lys Gly Arg Trp Val Leu Ile Thr Cys Cys Ala 20 25 30 Pro Gln Pro Pro Pro Pro Ile Thr Tyr Ser Leu Cys Gly Thr Lys Asn 35 40 45 Ile Lys Val Ala Lys Lys Val Val Lys Thr His Glu Pro Ala Ser Phe 50 55 60 Asn Leu Asn Val Thr Leu Lys Ser Ser Pro Asp Leu Leu Thr Tyr Phe 65 70 75 80 Cys Trp Ala Ser Ser Thr Ser Gly Ala His Val Asp Ser Ala Arg Leu 85 90 95 Gln Met His Trp Glu Leu Trp Ser Lys Pro Val Ser Glu Leu Arg Ala 100 105 110 Asn Phe Thr Leu Gln Asp Arg Gly Ala Gly Pro Arg Val Glu Met Ile 115 120 125 Cys Gln Ala Ser Ser Gly Ser Pro Pro Ile Thr Asn Ser Leu Ile Gly 130 135 140 Lys Asp Gly Gln Val His Leu Gln Gln Arg Pro Cys His Arg Gln Pro 145 150 155 160 Ala Asn Phe Ser Phe Leu Pro Ser Gln Thr Ser Asp Trp Phe Trp Cys 165 170 175 Gln Ala Ala Asn Asn Ala Asn Val Gln His Ser Ala Leu Thr Val Val 180 185 190 Pro Pro Gly Gly Asp Gln Lys Met Glu Asp Trp Gln Gly Pro Leu Glu 195 200 205 Ser Pro Ile Leu Ala Leu Pro Leu Tyr Arg Ser Thr Arg Arg Leu Ser 210 215 220 Glu Glu Glu Phe Gly Gly Phe Arg Ile Gly Asn Gly Glu 225 230 235 10124PRTHomo sapiens 10Val Ala Lys Lys Val Val Lys Thr His Glu Pro Ala Ser Phe Asn Leu 1 5 10 15 Asn Val Thr Leu Lys Ser Ser Pro Asp Leu Leu Thr Tyr Phe Cys Trp 20 25 30 Ala Ser Ser Thr Ser Gly Ala His Val Asp Ser Ala Arg Leu Gln Met 35 40 45 His Trp Glu Leu Trp Ser Lys Pro Val Ser Glu Leu Arg Ala Asn Phe 50 55 60 Thr Leu Gln Asp Arg Gly Ala Gly Pro Arg Val Glu Met Ile Cys Gln 65 70 75 80 Ala Ser Ser Gly Ser Pro Pro Ile Thr Asn Ser Leu Ile Gly Lys Asp 85 90 95 Gly Gln Val His Leu Gln Gln Arg Pro Cys His Arg Gln Pro Ala Asn 100 105 110 Phe Ser Phe Leu Pro Ser Gln Thr Ser Asp Trp Phe 115 120 11237PRTHomo sapiens 11Ala Gln Tyr Ser Ser Asp Arg Cys Ser Trp Lys Gly Ser Gly Leu Thr 1 5 10 15 His Glu Ala His Arg Lys Glu Val Glu Gln Val Tyr Leu Arg Cys Ala 20 25 30 Ala Gly Ala Val Glu Trp Met Tyr Pro Thr Gly Ala Leu Ile Val Asn 35 40 45 Leu Arg Pro Asn Thr Phe Ser Pro Ala Arg His Leu Thr Val Cys Ile 50 55 60 Arg Ser Phe Thr Asp Ser Ser Gly Ala Asn Ile Tyr Leu Glu Lys Thr 65 70 75 80 Gly Glu Leu Arg Leu Leu Val Pro Asp Gly Asp Gly Arg Pro Gly Arg 85 90 95 Val Gln Cys Phe Gly Leu Glu Gln Gly Gly Leu Phe Val Glu Ala Thr 100 105 110 Pro Gln Gln Asp Ile Gly Arg Arg Thr Thr Gly Phe Gln Tyr Glu Leu 115 120 125 Val Arg Arg His Arg Ala Ser Asp Leu His Glu Leu Ser Ala Pro Cys 130 135 140 Arg Pro Cys Ser Asp Thr Glu Val Leu Leu Ala Val Cys Thr Ser Asp 145 150 155 160 Phe Ala Val Arg Gly Ser Ile Gln Gln Val Thr His Glu Pro Glu Arg 165 170 175 Gln Asp Ser Ala Ile His Leu Arg Val Ser Arg Leu Tyr Arg Gln Lys 180 185 190 Ser Arg Val Phe Glu Pro Val Pro Glu Gly Asp Gly His Trp Gln Gly 195 200 205 Arg Val Arg Thr Leu Leu Glu Cys Gly Val Arg Pro Gly His Gly Asp 210 215 220 Phe Leu Phe Thr Gly His Met His Phe Gly Glu Ala Arg 225 230 235 1295PRTHomo sapiens 12Met Phe Gly Val Leu Glu Gly Ala Gln Ala Asn Ser Glu Asn Trp Ile 1 5 10 15 Ala Pro Ser Gly Pro Trp Ala Leu Gly Leu Trp Ser Ser Leu Tyr Phe 20 25 30 Leu Leu Phe Ser Thr Leu Glu Gly Arg Gly Gly Arg Val Leu Ser Gln 35 40 45 Ser Cys Ser Met Ala Val Ala Ala Ala Ser Trp Ile Ser Arg Glu Asn 50 55 60 Ala Arg Ser Val Lys Arg Ser Tyr Met Gln Ser Ser Pro Gln Arg Pro 65 70 75 80 Lys Glu Pro Arg Asn Gln Arg Thr Ser His Thr Thr Pro Val Cys 85 90 95 13205PRTHomo sapiens 13Ser Gly Phe Thr Ala Leu His Trp Ala Ala Lys Ser Gly Asp Gly Glu 1 5 10 15 Met Ala Leu Gln Leu Val Glu Val Ala Arg Arg Ser Gly Ala Pro Val 20 25 30 Asp Val Asn Ala Arg Ser His Gly Gly Tyr Thr Pro Leu His Leu Ala 35 40 45 Ala Leu His Gly His Glu Asp Ala Ala Val Leu Leu Val Val Arg Leu 50 55 60 Gly Ala Gln Val His Val Arg Asp His Ser Gly Arg Arg Ala Tyr Gln 65 70 75 80 Tyr Leu Arg Pro Gly Ser Ser Tyr Ala Leu Arg Arg Leu Leu Gly Asp 85 90 95 Pro Gly Leu Arg Gly Thr Thr Glu Pro Asp Ala Thr Gly Gly Gly Ser 100 105 110 Gly Ser Leu Ala Ala Arg Arg Pro Val Gln Val Ala Ala Thr Ile Leu 115 120 125 Ser Ser Thr Thr Ser Ala Phe Leu Gly Val Leu Ala Asp Asp Leu Met 130 135 140 Leu Gln Asp Leu Ala Arg Gly Leu Lys Lys Ser Ser Ser Phe Ser Lys 145 150 155 160 Phe Leu Ser Ala Ser Pro Met Ala Pro Arg Lys Lys Thr Lys Ile Arg 165 170 175 Gly Gly Leu Pro Ala Phe Ser Glu Ile Ser Arg Arg Pro Thr Pro Gly 180 185 190 Pro Leu Ala Gly Leu Val Pro Ser Phe Pro Pro Thr Thr 195 200 205 14125PRTHomo sapiens 14Ser Leu Ile Val Phe Met Glu Gln Val His Arg Gly Ile Lys Gly Leu 1 5 10 15 Val Arg Asp Ser His Gly Lys Gly Ile Pro Asn Ala Ile Ile Ser Val 20 25 30 Glu Gly Ile Asn His Asp Ile Arg Thr Ala Asn Asp Gly Asp Tyr Trp 35 40 45 Arg Leu Leu Asn Pro Gly Glu Tyr Val Val Thr Ala Lys Ala Glu Gly 50 55 60 Phe Thr Ala Ser Thr Lys Asn Cys Met Val Gly Tyr Asp Met Gly Ala 65 70 75 80 Thr Arg Cys Asp Phe Thr Leu Ser Lys Thr Asn Met Ala Arg Ile Arg 85 90 95 Glu Ile Met Glu Lys Phe Gly Lys Gln Pro Val Ser Leu Pro Ala Arg 100 105 110 Arg Leu Lys Leu Arg Gly Gln Lys Arg Arg Gln Arg Gly 115 120 125 1599PRTHomo sapiens 15Gly Ser Leu Ser Pro Thr Lys Tyr Asn Leu Leu Glu Leu Lys Glu Ser 1 5 10 15 Cys Ile Arg Asn Gln Asp Cys Glu Thr Gly Cys Cys Gln Arg Ala Pro 20 25 30 Asp Asn Cys Glu Ser His Cys Ala Glu Lys Gly Ser Glu Gly Ser Leu 35 40 45 Cys Gln Thr Gln Val Phe Phe Gly Gln Tyr Arg Ala Cys Pro Cys Leu 50 55 60 Arg Asn Leu Thr Cys Ile Tyr Ser Lys Asn Glu Lys Trp Leu Ser Ile 65 70 75 80 Ala Tyr Gly Arg Cys Gln Lys Ile Gly Arg Gln Lys Leu Ala Lys Lys 85 90 95 Met Phe Phe 1698PRTHomo sapiens 16Ser Gln Leu Glu Leu Ile Asp Leu Ser Ser Asn Pro Phe His Cys Asp 1 5 10 15 Cys Gln Leu Leu Pro Leu His Arg Trp Leu Thr Gly Leu Asn Leu Arg 20 25 30 Val Gly Ala Thr Cys Ala Thr Pro Pro Asn Ala Arg Gly Gln Arg Val 35 40 45 Lys Ala Ala Ala Ala Val Phe Glu Asp Cys Pro Gly Trp Ala Ala Arg 50 55 60 Lys Ala Lys Arg Thr Pro Ala Ser Arg Pro Ser Ala Arg Arg Thr Pro 65 70 75 80 Ile Lys Gly Arg Gln Cys Gly Ala Asp Lys Val Gly His Gly Ala Gly 85 90 95 Gly Val 17112PRTHomo sapiens 17Gly Glu Ala Gly Gly Ser Cys Leu Arg Trp Glu Pro His Cys Gln Gln 1 5 10 15 Pro Leu Pro Asp Arg Val Pro Ser Thr Ala Ile Leu Pro Pro Arg Leu 20 25 30 Asn Gly Pro Trp Ile Ser Thr Gly Cys Glu Val Arg Pro Gly Pro Glu 35 40 45 Phe Leu Thr Arg Ala Tyr Thr Phe Tyr Pro Ser Arg Leu Phe Arg Ala 50 55 60 His Gln Phe Tyr Tyr Glu Asp Pro Phe Cys Gly Glu Pro Ala His Ser 65 70 75 80 Leu Leu Val Lys Gly Lys Val Arg Leu Arg Arg Ala
Ser Trp Val Thr 85 90 95 Arg Gly Ala Thr Glu Ala Asp Tyr His Leu His Lys Val Gly Ile Val 100 105 110 1864PRTHomo sapiens 18Ala Thr Lys Leu Val Thr Cys Pro Ala Pro Arg Gln Phe Ala Val Gly 1 5 10 15 Ala Phe Thr Ala Ala Gly Arg Ala Trp Leu Phe Ala Pro Ser Leu Gly 20 25 30 Ala Ser Phe Ser Lys Leu Arg Ser Gln Gln Arg Ser Arg Asp Phe Arg 35 40 45 Gly Arg Leu Phe Leu Arg Ala Glu Arg Arg Ala Gly Gly Phe Thr Ser 50 55 60 19237PRTHomo sapiens 19Ser Ser Asn Leu Glu Gly Arg Thr Lys Ser Val Ile Arg Gln Thr Gly 1 5 10 15 Ser Ser Ala Glu Ile Thr Cys Asp Leu Ala Glu Gly Ser Thr Gly Tyr 20 25 30 Ile His Trp Tyr Leu His Gln Glu Gly Lys Ala Pro Gln Arg Leu Leu 35 40 45 Tyr Tyr Asp Ser Tyr Thr Ser Ser Val Val Leu Glu Ser Gly Ile Ser 50 55 60 Pro Gly Lys Tyr Asp Thr Tyr Gly Ser Thr Arg Lys Asn Leu Arg Met 65 70 75 80 Ile Leu Arg Asn Leu Ile Glu Asn Asp Ser Gly Val Tyr Tyr Cys Ala 85 90 95 Thr Trp Asp Gly His Ser Asp Ser Asp Pro Pro Tyr Thr Thr Leu Lys 100 105 110 Thr Cys Leu Val Ala Ala Ser Pro Arg Glu Glu Gly Met Arg Trp Ala 115 120 125 Leu Leu Val Leu Leu Ala Phe Leu Ser Pro Ile Pro Ala Pro Pro Thr 130 135 140 Val Phe Cys Ala Arg Asp His Phe Leu Val Glu Trp Asp Leu Ser Phe 145 150 155 160 Glu Arg Ile Phe Tyr Ser Phe Ser Ser Gly Pro Phe Pro Ser Lys Ala 165 170 175 Pro Glu Arg Lys Ala Cys Ser Gln Lys Ser Ser Asn Leu Glu Gly Arg 180 185 190 Met Lys Ser Val Thr Arg Pro Thr Gly Ser Ser Ala Glu Ile Thr Cys 195 200 205 Asp Leu Thr Val Ile Asn Ala Val Tyr Ile His Trp Tyr Leu Gln Gln 210 215 220 Glu Gly Lys Thr Pro Gln His Leu Leu His Tyr Asp Val 225 230 235 20145PRTHomo sapiens 20Ala Gly Val Thr Gln Thr Pro Lys Phe His Val Leu Lys Thr Gly Gln 1 5 10 15 Ser Met Thr Leu Leu Cys Ala Gln Asp Met Asn His Glu Tyr Met Tyr 20 25 30 Arg Tyr Arg Gln Asp Pro Gly Lys Gly Leu Arg Leu Ile Tyr Tyr Ser 35 40 45 Val Ala Ala Ala Leu Thr Asp Lys Gly Glu Val Pro Asn Gly Tyr Asn 50 55 60 Val Ser Arg Ser Asn Thr Glu Asp Phe Pro Leu Lys Leu Glu Ser Ala 65 70 75 80 Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Ser Thr 85 90 95 Ala Leu Gln Gly Cys Leu Leu Ser Ala His Lys Gly Lys Gly Arg Cys 100 105 110 Cys Pro Pro Pro Pro Pro Lys Thr Gln Gly Cys Pro Val Gln Arg Ser 115 120 125 Leu His Gln Glu Pro Trp Asn Pro Glu Trp Pro Gln Val Ala Arg Thr 130 135 140 Val 145 21143PRTHomo sapiens 21Ala Lys Val Thr Gln Thr Pro Gly His Leu Val Lys Gly Lys Gly Gln 1 5 10 15 Lys Thr Lys Met Asp Cys Thr Pro Glu Lys Gly His Thr Phe Val Tyr 20 25 30 Trp Tyr Gln Gln Asn Gln Asn Lys Glu Phe Met Leu Leu Ile Ser Phe 35 40 45 Gln Asn Glu Gln Val Leu Gln Glu Thr Glu Met His Lys Lys Arg Phe 50 55 60 Ser Ser Gln Cys Pro Lys Asn Ala Pro Cys Ser Leu Ala Ile Leu Ser 65 70 75 80 Ser Glu Pro Gly Asp Thr Ala Leu Tyr Leu Cys Ala Ser Ser Gln Ser 85 90 95 Thr Ala Leu Lys Cys Gln Phe Leu Leu Ala His Lys Leu Val Thr Asp 100 105 110 Pro Ala Gln Glu Ala Gly Asp Val Leu Gly Trp Lys Gly Val Thr Glu 115 120 125 Asn Asn Trp Ser Gln Leu Lys Pro Gln Cys Asn Leu Thr Gln Gly 130 135 140 2250PRTHomo sapiens 22Arg Asp Lys Met Arg Met Gln Arg Ile Lys Val Cys Glu Lys Arg Pro 1 5 10 15 Ser Ile Asp Leu Cys Ile His His Cys Ser Tyr Phe Gln Lys Cys Glu 20 25 30 Thr Asn Lys Ile Cys Cys Ser Ala Phe Cys Gly Asn Ile Cys Met Ser 35 40 45 Ile Leu 50 2355PRTHomo sapiens 23Pro Glu Arg Trp Phe Pro Gly Ser Cys His Val Phe Gly Gln Gly His 1 5 10 15 Gln Leu Phe His Ile Phe Leu Val Leu Cys Thr Leu Ala Gln Leu Glu 20 25 30 Ala Val Ala Leu Asp Tyr Glu Ala Arg Arg Pro Ile Tyr Glu Pro Leu 35 40 45 His Thr His Trp Pro His Asn 50 55 24237PRTHomo sapiens 24Gln Leu Leu Met Tyr Gln Gln His Thr Ser His Tyr Asp Leu Glu Arg 1 5 10 15 Lys Gly Gly Tyr Leu Met Leu Ser Phe Ile Asp Phe Cys Pro Phe Ser 20 25 30 Val Met Arg Leu Arg Ser Leu Pro Ser Pro Gln Arg Tyr Thr Arg Gln 35 40 45 Glu Arg Tyr Arg Ala Arg Pro Pro Arg Val Leu Glu Arg Ser Gly Phe 50 55 60 His Asn Glu Asn Ser Leu Ala Ile Tyr Gln Gly Leu Val Tyr Tyr Leu 65 70 75 80 Leu Trp Leu His Ser Val Tyr Asp Lys Pro Tyr Ala Asp Pro Val His 85 90 95 Asp Pro Thr Trp Arg Trp Trp Ala Asn Asn Lys Gln Asp Gln Asp Tyr 100 105 110 Tyr Phe Phe Leu Ala Ser Asn Trp Arg Ser Ala Gly Gly Val Ser Ile 115 120 125 Glu Met Asp Ser Tyr Glu Lys Ile Tyr Asn Leu Glu Ser Ala Tyr Glu 130 135 140 Leu Pro Glu Arg Ile Phe Leu Asp Lys Gly Thr Glu Tyr Ser Phe Ala 145 150 155 160 Ile Phe Leu Ser Ala Gln Gly His Ser Phe Arg Thr Gln Ser Glu Leu 165 170 175 Gly Thr Ala Phe Gln Leu His Ser Gln Val Asp Val Gly Val Val Leu 180 185 190 Ala Asp Pro Gly Cys Ile Glu Ala Ser Val Lys Gln Glu Val Leu Ile 195 200 205 Asn Arg Asn Ser Val Leu Phe Ser Ile Thr Leu Lys Asp Lys Lys Leu 210 215 220 Cys Tyr Asp Gln Gly Ile Ser Gly His His Leu Met Glu 225 230 235 25140PRTHomo sapiens 25Val Val Glu Glu Leu Lys Leu Ser His Asn Pro Leu Lys Ser Ile Pro 1 5 10 15 Asp Asn Ala Phe Gln Ser Phe Gly Arg Tyr Leu Glu Thr Leu Trp Leu 20 25 30 Asp Asn Thr Asn Leu Glu Lys Phe Ser Asp Gly Ala Phe Leu Gly Val 35 40 45 Thr Thr Leu Lys His Val His Leu Glu Asn Asn Arg Leu Asn Gln Leu 50 55 60 Pro Ser Asn Phe Pro Phe Asp Ser Leu Glu Thr Leu Ala Leu Thr Asn 65 70 75 80 Asn Pro Trp Lys Cys Thr Cys Gln Leu Arg Gly Leu Arg Arg Trp Leu 85 90 95 Glu Ala Lys Ala Ser Arg Pro Asp Ala Thr Cys Ala Ser Pro Ala Lys 100 105 110 Phe Lys Gly Gln His Ile Arg Asp Thr Asp Ala Phe Arg Ser Cys Lys 115 120 125 Phe Pro Thr Lys Arg Ser Lys Lys Ala Gly Arg His 130 135 140
Patent applications in class By measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Patent applications in all subclasses By measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
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subset: commoncrawl


text:
Inter President Massimo Moratti is convinced he made the right decision in hiring Coach Walter Mazzarri – despite the club's recent struggles.
After a fine start to the season, the Nerazzurri have only collected two points from their games against Cagliari, Roma and Torino.
"Let's say we've not exactly had the rub of the green in the last three games, but we've also made mistakes of course," Moratti told Inter Channel.
"But I think these matches have served as great experience for Mazzarri because they've allowed him to go into greater depth on the team, players and tactical systems.
"Mazzarri is a very reactive person, he's very capable and he's in a new environment. He also needs to gradually find his feet.
"The players appreciate him a lot because he's got a phenomenal work ethic.
"He certainly has the ability to talk to everyone, he doesn't do it because he has to, but because that's who he is.
"He's a very capable Coach, I think he'll be able to consolidate all this with performances that make us happy and that we deserve."
Mazzarri, who left Napoli after leading them to a second-place finish, arrived at the club in the summer to replace Andrea Stramaccioni.
The Beneamata will be facing Verona next. "I think everyone's been surprised by their brilliant start," added Moratti.
"A team that comes up from Serie B and has all this success... I can't remember many."
See the latest Serie A predictions and betting tips with Eurotips.co.uk


text:
UN observers leave Syria: too much violence to observe
The UN mission on their way out of Syria
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The United Nation's troubled observer mission to Syria has officially ended after being recalled amid escalating violence as world powers fail to agree how to end months of bloodshed in the country.
The mandate of the U.N. Supervision Mission in Syria (UNSMIS) expired at midnight Sunday after a roughly four-month deployment in which its work was hobbled by growing unrest that has cost tens of thousands of lives.
Created after a U.N. Security Council resolution in April, a team of some 300 truce monitors was progressively deployed into Syria as part of U.N.-Arab League envoy Kofi Annan's six-point plan to end the conflict.
It was suspended in June and numbers cut back because of the mounting violence, as both sides violated a ceasefire that was meant to have been the cornerstone of Annan's plan.
The departing U.N. observer mission chief on Saturday accused both Syrian army and rebel forces of failing to protect civilians.
"Both parties have obligations under international humanitarian law to make sure that civilians are protected," General Babacar Gaye, head of the U.N. Supervision Mission in Syria, told reporters in Damascus.
"These obligations have not been respected."
Annan, a former U.N. secretary general, steps down as international envoy on Syria at the end of this month after complaining about a lack of international support for his six-month campaign to make President Bashar al-Assad and opposition fighters end their hostilities.
The United Nations plans to maintain a political liaison office in Damascus to support the mediation efforts of his successor, veteran Algerian diplomat Lakhdar al-Ibrahim.
Officials have said the liaison office would probably be made up of between 20 and 30 people, including political, humanitarian and military experts.
Continued violence
Early Monday, in the second day of the Muslim occasion of Eid al-Fitr, clashes between Free Syrian Army and Assad's forces marred al-Tadhamun neighborhood in Damascus, Syrian Revolution General Commission reported.
In the capital, smoke was also seen billowing from al-Maza airport, the Local Coordination Committees reported.
LCC said more than 170 people were killed across Syria on Saturday.
The Syrian Media Center, meanwhile, reported on Monday of people killed and wounded after rocket shelling on the southern city of Deraa al-Balad.
Ahmed al-Darawi a representative from the center in Deraa described the situation in Deraa al-Balad as "miserable."
Asked about the expected efforts from Ibrahimi, Darawi said the new U.N. envoy cannot fix what Assad has spoiled.
Meanwhile, Ibrahimi denied that he said that it will be early to talk about the stepping down of President Assad. He also rebuffed requests from the Syrian opposition for him to apologize for the alleged statement, saying that it will not only be not suitable for him but also for the opposition to do so.
What began in March 2011 as a peaceful uprising demanding the fall of Assad's regime has grown into a bloody insurgency, after the army and security forces launched a major crackdown across the country.
More than 23,000 people have been killed, according to the Britain-based Syrian Observatory for Human Rights, while the U.N. puts the death toll at 17,000 people.
Has the UN done a good job in Syria? Should they stay? Tell us what you think below.


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Religion briefs
Published 7:00 pm, Wednesday, April 8, 2009
Churches unite
Five area churches will come together during Holy Week to host a variety of services meant to unite the parishioners who attend different services in their neighborhoods and to open the doors to any newcomers.
The Rev. Robert Barndt, pastor of Midland Lutheran Church, said during the Easter season, pastors and parisioners seem more willing to come together to celebrate Christ's death and resurrection.
"We are more open to letting down our guard this time of year," he said.
Three of the churches started the tradition of celebrating Holy Week together about 10 years ago, and since then the group has grown to include two others: Church on the Journey, Memorial Christian Church, Midland Lutheran Church, St. Luke's United Methodist Church and Trinity Presbyterian Church.
The churches will host a community lunch at 11:45 a.m. Monday through Wednesday, followed by a short devotional service. The congregations will meet Monday at St. Luke's, Tuesday at Memorial Christian Church and Wednesday at Midland Lutheran Church, where the lunch will be hosted by Church on the Journey.
The week, Barndt said, is a good time for visitors to attend because "we don't know who's a member and who isn't. We're not looking for members just to share the message," he said.
Holy Week
n St. Luke's United Methodist Church: Easter cantata for 11 a.m. Sunday, Maundy Thursday Service for 6:30 p.m. April 9 and Easter celebration for 8:30 a.m. and 10:50 a.m. April 12.
n St. Nicholas' Episcopal Church: Holy Eucharist and Palm Liturgy, 9 a.m. Sunday at the Parish Hall; Stations of the Cross, 5:30 p.m. Monday and Tuesday; Holy Eucharist and healing service, 5:30 p.m. Wednesday in the chapel; Maundy Thursday Liturgy, 8 p.m. Thursday; Good Friday Liturgy, 12:05 p.m. Friday; Easter vigil, 9 p.m. April 11; and Easter Communion Service, 9 a.m. April 12.
n Redeemer Lutheran Church of Joy, 824 E. 18th St., Odessa: Good Friday tenebrae service (service of darkness), 7 p.m. Friday; and worship service, 10:30 a.m. April 12 (preceded by breakfast at 9 a.m.).
n Greater Mount Zion Baptist Church: presentations on "The Seven Last Words of Jesus Christ on the Cross" 7 p.m. Wednesday through Friday; and Easter sunrise service, 6 a.m. April 12.
n Life Unlimited Church, 4224 NE Loop 338, Odessa: Palm Sunday drama, 10:15 a.m. Sunday; and Easter pageant and drama, 10:15 a.m. April 12.
Dave and Barb Anderson are to perform 6 p.m. Sunday at Grace Lutheran Church. During the concert, the couple will share their experiences of being rescued, along with five other people, after a plane crash in the Bering Sea in 1993. Donations will be taken for Shepherd's Canyon Retreat, a counseling and retreat center the Andersons are planning near Phoenix.
Financial classes
Believersway Church, 4400 N. Midland Drive, is planning Financial Peace University beginning Monday at 6:30 p.m. For more information or to register, contact Mike Beggs at 556-6359 or firstname.lastname@example.com.
Economic discussion
Four local churches will be broadcasting a town-hall meeting with Dave Ramsey, set for 7 p.m. April 23.
Ramsey, a nationally syndicated radio talk show host and New York Times best-selling author, will be talkng about the economic crisis.
The churches showing the free event are:
n Christ Church Midland. For more information or to register, contact Mark Kratzer at 818-2221 or firstname.lastname@example.com for more information or to register.
n True-Lite Christian Fellowship. For more information or to register, contact Roy Smith at 687-3077 or email@example.com
n Crestview Baptist Church. For more information or to register, contact Becky McCraney at 352-7365 or email@example.com.
n First United Methodist Church. For more information or to register, contact Daniel Harris at 682-370 or firstname.lastname@example.com.
Open house
Christian Science Reading Room, 4400 N. Big Spring St., is to host an open house 11 a..m.-3 p.m. April 16 and 5-6 p.m. April 18-19. The open house is planned in conjunction with the Christian Science Monitor being converted to a weekly newspaper. For more information, call 682-8816.
Lorenzo Rodriguez of Miami Shores, Fla., a member of the Christian Science Board of Leadership, is to speak April 18 and 19 in Midland. He will present a talk on prayer-based solutions to problems at 3:30 p.m. April 18 at Martin Luther King Community Center and "The True Brotherhood of Man" at 3:30 p.m. April 19 at First Church of Christ Scientist.
Opera singer
Mezzo-soprano, Luretta Bybee, a Lee High School graduate, will sing at First United Methodist Church at the 8:30 and 10:45 a.m. services on April 12. Bybee, the daughter of FUMC members Cecil and Helen Bybee, will sing a duet with her daughter, Emma Grimsley 17.
Easter play
First Assembly Church of God is to present the drama "You Are My Victory" 6 p.m. April 11 and 10:30 a.m. April 12.


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Chelsea star Eden Hazard knows he needs to step up his World Cup form for Belgium.
Hazard is expected to start for Belgium in their World Cup round of 16 clash with the USA.
"In this World Cup, I have yet to play a great match, even if I had good moves which were decisive," Hazard acknowledged.
"They are asking me to be decisive, and it has been tough."
For the big stories and the banter follow us on Twitter: @tribalfootball


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Those who did so used their medical training not to care for patients, but to abet their abuse.
To view these nudes is not quite to abet evil, but it is to undermine decency.
Yet, far from nudging Rajapaksa toward greater accountability, their presence in Sri Lanka is likely only to abet his rise.
He was there to aid and abet when Wade got hot in the fourth quarter.
Our political leaders need to demand that their colleague step down immediately, not aid and abet the barbarian criminal.
He has killed the king's men; and if the baron should aid and abet, he will lose his castle and land.
Lucy smiled at the bare-faced fraud and hastened to abet it.
She had striven to aid and abet this distinguished and worthy gentleman in his suit.
And you will not abet revolutionary measures if you get into Parliament?
How does abet differ from incite and instigate as to the time of the action?
late 14c. (implied in abetting), from Old French abeter "to bait, to harass with dogs," literally "to cause to bite," from a- "to" (see ad-) + beter "to bait," from a Germanic source, perhaps Low Franconian betan "incite," or Old Norse beita "cause to bite," from Proto-Germanic *baitjan, from PIE root *bheid- "to split" (see fissure). Related: Abetted; abetting.


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If the little house perched on a pedestal in Bruce Blaisdell's yard had been built for birds, it would provide a spacious, comfy home to some lucky robins.
The glass door on the front isn't meant to keep predators out, though. Instead, it swings open freely, and regularly, offering entertainment options for neighborhood kids looking to mentally escape the confines of their own homes after school – without turning on their Xbox or Playstation machines.
They walk by the retired teacher's Marshall Street home on their way to and from Jefferson Elementary. His "Little Library" has a note on the window that says the kids are free to take a book or leave one.
There are adult books in the little house, too, but Blaisdell said he's added more children's books recently because he's noticed kids are using his library the most. As an example, there were several titles from the popular "Baby Sitters Club" series and "Boxcar Children" series. Adults could find books such as "Trinity" for themselves, and "Barnyard Dance" was waiting for the parent of a lucky toddler.
"Reading is probably the best trip in the world, isn't it?" Blaisdell said. "I've had people stop and knock on the door and say, 'What a great idea.' Anytime you can encourage someone to read a book, it's a good thing."
He got the idea for the fancy book bin while visiting his daughter in Faribault. She brought him to Dale Smith's house and showed him the Little Free Library he had built on the corner of his yard.
Blaisdell said he became more intrigued after his daughter told him the small neighborhood book exchanges were popping up all over the country.
"Once I found that out, I said let's get on the bandwagon," he said.
With his tongue in his cheek, Todd Bol of Hudson, Wis., jokingly refers to Blaisdell's creation as a rogue Little Free Library because it doesn't have an official sign. Bol is the guy credited with starting the idea, which has reached far beyond Faribault and Mankato. He's offered to have an official sign made for anyone who is willing to build a library.
There are now hundreds of Little Free Libraries in the United States and overseas. There's also an official Little Free Library website.
"I started it about two years ago," Bol said. "I just built one in my yard and people responded to it so positively during a garage sale.
"I built it to slow down traffic along the St. Croix River and to honor my mom (June Bol). I had no real intention of building more."
After seeing so many people "light up" during that garage sale, Bol contacted his friend, Rick Brooks, in Madison, Wis., and worked out a plan to put up little libraries in that city. There were more than 70 sites in the Madison area at last count.
Fancy-looking versions of the libraries have sprouted all over Louisiana since a woman in New Orleans put one up and drew the attention of state media. Something similar happened in northern Minnesota when a Detroit Lakes woman's library was the subject of a news feature.
There have been calls from reporters as far away as Sweden and Israel, Bol said.
"I think its explosion is yet to come," he said. "I made my 725th sign the other day. Every day I get an email about a new one being built. You have to figure, come spring time or summer, there will be two or three going up every day.
"One person in the community starts the seed, then more people start building and growing the idea. It has a way of pulling a community together."
Those seeds are already spreading in Mankato. Blaisdell has been getting some help from Mark Hustad, owner of Once Read Book Exchange on Front Street in downtown Mankato.
Blaisdell went into the store recently looking for children's books to add to his collection. He used his phone to show Hustad a picture of his Little Library, which is painted to match his house and includes solar-powered lights.
Hustad told him the library already had caught his attention during a Sunday walk, then gave him a great deal on a bunch of children's books that were about to be recycled.
For more information about the libraries, go to www.LittleFreeLibrary.org or contact Bol at firstname.lastname@example.com .