L Meheus

An updated two-dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies.

A standard two‐dimensional (2‐D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2‐D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol‐lowering drugs and a high‐cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2‐D gel system (the Iso‐Dalt® system), it can be directly related to an expanding body of work in other laboratories.

Rational Design of a Multiepitope Vaccine Encoding T-Lymphocyte Epitopes for Treatment of Chronic Hepatitis B Virus Infections

ABSTRACT Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2bxd (BALB/c × C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.

Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization — mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry

Optimal application of biological mass spectrometry (MS) in combination with two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2‐D PAGE protein spots in a human CSF pool using spot co‐localization, N‐terminal sequencing, matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) and nanoliquid chromatography‐electrospray ionization‐time of flight‐mass spectrometry (nanoLC‐ESI‐TOF‐MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2‐D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N‐terminal sequence analysis and MALDI‐MS peptide mass fingerprint analysis of both silver‐ and Coomassie Brilliant Blue (CBB) G‐250‐stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2‐D ExPASy map: complement C3 α‐chain (1321—1663), complement factor B, cystatin C, calgranulin A, hemoglobin β‐chain, and β‐2‐microglobulin. It was clear that MALDI‐MS identification from CBB G‐250‐stained, rather than from silver‐stained, spots was more successful. In cases where no N‐terminal sequence and/or no clear MALDI‐MS result was available, nanoLC‐ESI‐TOF‐MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low‐abundant proteins on 2‐D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2‐D PAGE of the fractionated CSF pool showed various new silver‐stainable protein spots, of which four were identified by nanoLC‐ESI‐TOF‐MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.

Detection and identification of antinuclear antibodies (ANA) in a large and consecutive cohort of serum samples referred for ANA testing

OBJECTIVE To provide data on (a) the probability of detecting antinuclear antibodies (ANA) in a large and consecutive cohort of serum samples referred for ANA testing and (b) the probability of detecting more specific antinuclear reactivities (anti-DNA and anti-extractable nuclear antigens (anti-ENA)) in serum samples with a positive screening test (indirect immunofluorescence on HEp-2 cells). METHODS Serum samples from 10 550 consecutive patients sent to the laboratory for ANA detection were analysed. In ANA positive serum samples (23.5% of referred serum samples), ANA were identified by indirect immunofluorescence on Crithidia, by immunodiffusion, and by line immunoassay. Because anti-SSA antibodies were the most frequently identified ANA, sensitively detected by line immunoassay, additional immunoassays were developed to confirm the specificity of the line immunoassay result. RESULTS At least one fine reactivity could be identified in 21.1% of ANA positive serum samples: anti-dsDNA in 3.2%; anti-ENA (anti-SSA 10.5%, anti-SSB 6.7%, anti-RNP 2.7%, anti-Sm 1.8%, anti-Scl70 1.2%, anti-Jo-1 0.2%) in 15.8%, rRNP and anti-Cenp-B in respectively 0.5% and 4.0%. Multiple reactivities were found in 7.9%. For anti-ENA antibodies, line immunoassay was more sensitive than immunodiffusion (15.4%v 7.7%; p<0.0001). The sensitive detection of anti-SSA antibodies by line immunoassay was confirmed by additional assays. CONCLUSIONS The data from this analysis are useful in estimating the probabilities of detecting specific ANA. Line immunoassay was shown to be a sensitive test for the detection of anti-ENA antibodies.

Anti-Ro52 reactivity is an independent and additional serum marker in connective tissue disease

Objective: To determine whether anti-Ro52 is an independent serum marker in connective tissue disease. Methods: Over a two year period, 1727 consecutive antinuclear antibody (ANA) positive serum samples were analysed in parallel by double immunodiffusion with thymus/spleen nuclear extract and by line immunoassay with recombinant Ro52, recombinant La/SSB, and natural Ro60. Sera that were only reactive towards Ro52 were further analysed by a variety of additional anti-SSA/Ro detection methods and by specific anti-Ro52 and anti-Ro60 assays. Natural purified SSA/Ro was analysed by immunoblot and protein sequencing. Results: Analysis of natural purified SSA/Ro (Immunovision, Springdale, AR) showed only Ro60 and no immunoreactive Ro52. Consequently, assays based on this substrate only identify sera with anti-Ro60 reactivity. Twenty serum samples showed anti-Ro52 without anti-Ro60 and anti-SSB/La on line immunoassay. By additional testing, 2/20 sera were found positive for anti-Ro60 reactivity. The remaining 18 sera were not identified by any of the classical anti-SSA/Ro assays and were considered to be reactive only with Ro52 and not with Ro60. This anti-Ro52 reactivity was confirmed by natural and recombinant Ro52 in 16/18 cases. 12/18 sera corresponded to connective tissue diseases. Conclusion: Anti-Ro52 positive sera without any evidence of anti-Ro60 and anti-La/SSB reactivity can be considered as an independent group that is systematically missed by classical anti-SSA/Ro detection methods owing to a bias towards anti-Ro60 reactivity. The anti-Ro52 sera are precipitin negative, not retrieved by SSA/Ro enzyme linked immunosorbent assays (ELISAs) based on natural SSA/Ro, and show no specific ANA fluorescence staining pattern. These findings together with the clinical data indicate that anti-Ro52 should be considered as an additional and independent serum marker.

Diagnostic associations in a large and consecutively identified population positive for anti-SSA and/or anti-SSB: the range of associated diseases differs according to the detailed serotype

Objective: To determine the diagnostic distribution in a consecutive anti-SSA and/or anti-SSB positive population. Methods: A total of 15 937 serum samples from 10 550 consecutive patients were analysed for antinuclear antibodies (ANAs) on HEp-2 cells. Serum samples positive for ANAs were analysed by immunodiffusion and line immunoassay with recombinant SSA-Ro52, natural SSA-Ro60, and recombinant SSB. Results: Among ANA positive patients in whom clinical information was available, 181 consecutive patients with anti-SSA and/or anti-SSB antibodies were identified, Disease associations were systemic lupus erythematosus (SLE) (45.3%), primary Sjögrens syndrome (pSS) (14.4%), scleroderma (8.8%), RA (7.7%), cutaneous lupus (7.7%), and dermatomyositis (2.2%). The ratio of diagnoses differed according to the anti-SSA/anti-SSB serotype. Scleroderma and dermatomyositis were enriched among mono-Ro52 reactive serum samples (34.2% and 10.5% respectively). Single reactivity towards Ro60 or anti-Ro60 with anti-Ro52 predisposed for SLE (80.0% and 52.2% respectively). Triple reactivity towards Ro52, Ro60, and SSB was primarily linked with SLE (55.8%) followed by pSS (20.9%). Anti-SSA on immunodiffusion increased the chance for SLE (62.8%), whereas isolated anti-SSB reactivity on immunodiffusion was less indicative for SLE (14.3%) and predisposed more for cutaneous lupus (23.8%) and pSS (33.3%). Conclusion: The diagnostic range associated with anti-SSA or anti-SSB reactivity differs significantly according to the detailed serotype defined by line immunoassay and immunodiffusion.

CHANGES IN THE LIVER PROTEIN PATTERN OF FEMALE WISTAR RATS TREATED WITH THE HYPOGLYCEMIC AGENT SDZ PGU 693

SDZ PGU 693 acts as a hypoglycemic agent by stimulating glucose utilisation in insulin-sensitive peripheral tissues, such as skeletal muscle and fat. In a 28 day toxicity study the compound was found to induce hepatocellular hypertrophy in Wistar rats treated with 300 mg/kg/day. To gain insights into the pathomechanism of these alterations, aliquots of liver samples from control and treated female Wistar rats were separated by two-dimensional protein gel electrophoresis and the digitized images of the protein patterns were searched for protein abundance changes. Significant treatment-related quantitative changes (P < 0.001) were found in 29 liver proteins. Major increases were observed in several microsomal proteins, including NADPH cytochrome P-450 reductase, cytochrome b5 and serine protease inhibitor. The changes in the cytochrome related enzymes, both known co-factors of the P-450 enzyme system, strongly suggest that SDZ PGU 693 induces microsomal proliferation and induction of the P-450 enzyme system. Decreases were observed in a series of mitochondrial proteins, such as F1ATPase-delta subunit and ornithine aminotransferase precursor as well as in several cytosolic proteins such as the liver fatty acid binding protein, arylsulfotransferase and the senescence marker protein-30. The changes in F1ATPase-delta subunit and liver fatty acid binding protein together suggest a down-regulation of the mitochondrial liver fatty acid metabolism, likely reflecting the pharmacological action of the compound. These results show that SDZ PGU 693 produces a complex pattern of gene expression changes which give insights into the molecular mechanisms of both its pharmacological action and a toxic response.

Clinical optimization and multicenter validation of antigen-specific cut-off values on the INNO-LIAr ANA Update for the detection of autoantibodies in connective tissue disorders

OBJECTIVES The INNO-LIA ANA Update is a qualitative multiparameter line immunoassay for detection of autoantibodies to several different antigens associated with connective tissue disorders. We sought to optimize and validate the cut-off values for its antigen-specific components: SmB, SmD, RNP-70k, RNP-A, RNP-C, SSA/Ro52, SSA/Ro60, SSB/La, Cenp-B, Topo-I, Jo-1, ribosomal P, and histones. Our aim was to achieve 98% specificity for each of the markers, with respect to differential disease controls, while maintaining sensitivity. METHODS For optimization, the cut-off value of the different antigen lines was fixed to achieve this specificity using an in-house set of 955 patient samples. Specificity was validated at multiple sites using a different set of 330 samples obtained from 158 apparently healthy blood donors, 100 patients with a variety of infections, 20 each with Wegeners granulomatosis, inflammatory bowel disease, and primary antiphospholipid syndrome, and 12 with psoriatic arthritis. Sensitivity was evaluated, using this optimized cut-off control, in 147 patients with scleroderma, 93 with Sjögrens disease, 40 with systemic lupus erythematosus, 40 with rheumatoid arthritis, 39 with mixed connective tissue disease, and 19 with polymyositis. Sensitivity and specificity of the INNO-LIA ANA Update were determined using the clinical diagnosis as reference. RESULTS The optimized cut-off values resulted in a specificity 98% or more for all LIA markers except one (histones 97.8%) in the validation set of 330 samples. The sensitivity for each marker tested in 378 samples from the target patient groups was comparable to that reported in the literature. CONCLUSION The INNO-LIA ANA Update shows uniformly high specificities combined with sensitivities very similar to those of reference assays, in a single test format.

Presence of anti-RNP-A and anti-RNP-C antibodies is inversely associated with renal symptoms of systemic lupus erythematosus

1 Rheumatoid arthritis — class prediction by autoreactivity profiles R Bergholz1, F Schumann1, S Behrens1, U Ungethüm1, G Valet2, WA Schmidt3, GR Burmester1, JM Engel4, WJ van Venrooij5, G Steiner6, S Bläß1 1Department of Rheumatology & Clinical Immunology, Charité University Clinic, Berlin, Germany 2MPI Biochemistry, Munich, Germany 3Clinic for Rheumatology Berlin Buch, Berlin, Germany 4Rheumaklinik, Bad Liebenwerda, Germany 5Department of Biochemistry, University of Nijmegen, The Netherlands 6Divison of Rhematology, Department Internal Medicine III, Vienna General Hospital, Austria Arthritis Res Ther 2003, 5 (suppl 1):1

Detection of rheumatoid arthritis-specific anti-filaggrin antibodies by line immunoassay shows complementarity to RF and corresponds to the AFA-blot using natural antigen

Anti-filaggrin autoantibodies (AFA) are highly specific markers for rheumatoid arthritis (RA) and can be detected by immunoblotting using human epidermal protein extracts. Furthermore, it was demonstrated that citrullination of the filaggrin epitopes is crucial for epitope recognition and that citrullinated peptides are also recognized by these specific autoantibodies. Based on these data, a line immunoassay (LIA) was developed containing as individual markers in vitro citrullinated recombinant filaggrin and two citrullinated synthetic peptides. Firstly, a comparison was made between this prototype LIA and the AFA blot using natural filaggrin. A blind serum panel consisting of 25 early RA, 25 longstanding RA, and 25 disease controls was selected. Results showed a similar performance of both tests at a specificity level of 95%, while the LIA proved significantly better (P = 0.035) than the AFA blot at 99% specificity. At the latter specificity level, 2 out of 17 RF negative samples were retrieved on LIA but not on Western blot. The LIA was further evaluated on sera obtained from 335 RA patients and 254 patients with non-RA rheumatological disorders in a retrospective study. The overall sensitivity of the LIA including three markers (LIA3) was 65.1% versus 61.8% if only the reactivity towards the citrullinated peptides was considered (LIA2). The specificity of LIA3 was 97.6% versus 98.4% for LIA2, which correlates with an estimated positive predictive value (PPV) of 87.3% for LIA3 and 90.7% for LIA2. Thirty-seven percent (30/81) of the RF-negative RA samples proved LIA2-positive, while 52 out of 55 RF positive control samples were negative for anti-filaggrin. Higher specificity and sensitivity was obtained for LIA2 in comparison with anti-RA33 immunoblot, whereas good agreement could be observed with anti-keratin antibody (AKA) testing. In conclusion, anti-filaggrin autoantibodies can be readily detected by a LIA test based on citrullinated peptides, resulting in a high specificity and hence high PPV for RA. The assay can serve as a user-friendly alternative for AKA immunofluorescent and immunoblot techniques. Together with the RF complementarity, this test provides a valuable tool in the differential diagnosis of RA.